Climate change increases toxic cadmium loads more than nutritional metals in spinach

Climate change increases toxic cadmium loads more than nutritional metals in spinach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Climate change increases toxic cadmium loads more than nutritional metals in spinach Aleksandra Pieńkowska, Alexandra Glöckle, Natalia Sánchez, Shitalben Khadela, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5947512/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In addition to food quantity, food quality is paramount for meeting the demands of a growing global population. Food quality encompasses both nutritional and contaminant contents, yet their transfer within soil-crop systems remains poorly understood under impending climate change. This greenhouse study is the first to demonstrate that future climatic conditions increase the transfer of metals from oxic soils to crops, showcased for four soil-spinach variety combinations ( Spinacia oleracea ). Future conditions raised harmful metal cadmium levels in edible spinach tissues by 26–54%. In contrast, changes in micronutrient (Zn, Mn, Mg) contents were inconsistent and dependent on the specific soil-spinach combination. Climate-induced shifts in soil carbon composition and bacterial communities were linked to greater soil Cd phytoavailability, enhancing Cd transfer from soil to roots. These findings suggest that while spinach's nutritional values may remain stable, future conditions could lead to higher metal contaminants levels in edible tissues. crops heavy metal nutrients elevated temperature elevated CO2 soil moisture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The quality of food is intricately linked to the presence and amounts of micronutrients and contaminants, holding relevance for human health 1 . Foods rich in essential micronutrients like zinc (Zn), magnesium (Mg), or manganese (Mn) are vital for supporting various physiological functions including immune function, fertility, muscles and bone growth 2 . However, current global food systems face challenges in providing adequate micronutrients with, for example, 17% of the global population deficient in dietary Zn 3 . The intensification of agriculture since the 1960s has led to higher biomass yields but caused micronutrient dilution in crops 4 . Additionally, non-essential metals such as cadmium (Cd), can accumulate in crops, posing significant health risks to consumers, including carcinogenic effects and damage to the kidneys and skeletal system 5 . Nutritional and contaminant metals, occur naturally in agricultural soils, but their concentrations and mobility are influenced by human activities such as farming, industrial operations, and vehicle emissions 6 , 7 . In Europe and the US, around 75% of topsoils contain moderate Cd levels, with some soils exceeding 1 mg Cd kg⁻¹ dry soil still used for food production 8 , 9 . Recently it was demonstrated that future climatic conditions may increase Cd mobility in agricultural soils 10 . 4°C higher daytime temperatures, doubled atmospheric CO 2 and slightly lower soil moisture caused two times higher Cd concentrations in the pore water of these soils, which even transferred into the mobile fraction of the soil approximated with 0.01 M CaCl 2 extractions. This effect is strongest in soils with a pH 7 showed no significant shifts due to Cd binding with carbonate minerals 10 . The observed increase in the concentration of Cd in the porewater due to climate change raises concerns about the potential transfer of this harmful metal into crops grown in oxic soil systems, posing a threat to human nutrition. This phenomenon has already been demonstrated for rice grown in flooded soils under greenhouse conditions, where future climatic conditions increased the bioavailability of the most prominent contaminant in rice systems, arsenic, ultimately decreasing rice yields and increasing regulatory inorganic grain arsenic limits 12 . Such links between climate change, metallic contaminant, or micronutrient phytoavailability in oxic soils - approximately 90% of agricultural soil 13 -remain understudied. Elevated atmospheric CO 2 and temperature have differential effects on plant growth and performance. Increasing atmospheric CO 2 stimulates the photosynthetic rates, increasing water and nutrient use efficiency and allocation of more photosynthetic product to the root, ultimately resulting in greater biomass and increased yields 14 . As temperature rises, photosynthetic rates and yields increase up to the plant's optimum, but excessive heat can reduce yields and offset the positive effects of elevated CO₂ 15 , 16 . Here, we examine for the first time the climate change induced transfer of harmful Cd and nutritional Zn into spinach, a leafy vegetable contributing 32 million tons annually to global food production 17 , which is grown on oxic soils and known for accumulating Cd in edible parts 18 . Our findings shed light on the potential risks posed by metal contamination to crops in the future and highlight the importance of mitigating these risks for food security and quality. For this study, we utilized four geochemically diverse agricultural soils from different areas in Germany and four commonly cropped spinach varieties to examine the potential compounding impacts of a changing climate on soil metal fate. Natural soil Cd and Zn concentrations ranged from background to contaminated 8 , 9 (Figure S1A). For today’s climate variables, we examined 20°C midday temperatures, ambient atmospheric CO 2 , and 45% soil water holding capacity, which represent climatic conditions commonly used for spring and fall cultivation of these spinach varieties according to suppliers’ recommendation. Future climatic conditions applied in this study are compared to today's baseline and included a 3.4°C temperature rise, a 290 ppm v atmospheric CO₂ increase, and a 2-percentage-point decrease in soil moisture relative to water-holding capacity. These conditions align with the projected climate scenario under Shared Socioeconomic Pathway SSP2–4.5 19 , identified as the most likely scenario for the year 2100 relative to today 20 , and a SSP4-8.5 scenario relative to pre-industrial times. RESULTS Determinants of edible spinach yield and quality Cadmium accumulated in edible leaves of the Schlunzig-Lazio, Großschirma-Butterflay, Tübingen-Metador, Schladebach-Corvair combinations to 1.76 ± 0.11, 3.58 ± 0.28, 0.27 ± 0.01 and 0.39 ± 0.04 µg Cd g − 1 dry leaf, respectively, under today’s climatic conditions (Fig. 1 A). A shift to future climatic conditions statistically increased leaf Cd for Schlunzig-Lazio by 54% to 2.71 ± 0.19 µg g − 1 , Großschirma-Butterflay by 27% to 4.55 ± 0.37 µg g − 1 , Tübingen-Metador by 30% to 0.35 ± 0.02 µg g − 1 , and Schladebach-Corvair by 26% to 0.53 ± 0.03 µg Cd g − 1 dry leaf. Root Cd concentrations were 1.1-6.9-times higher than in leaves under today's climatic conditions, depending on the soil-spinach combination. Under future climatic conditions, root Cd increased by 26–54%, with a significant increase for the Großschirma-Butterflay combination (Table S1A). The micronutrient Zn accumulated to 75 ± 4, 92 ± 14, 67 ± 3 and 53 ± 5 µg Zn g − 1 dry leaf in edible leaves of the Schlunzig-Lazio, Großschirma-Butterflay, Tübingen-Metador, Schladebach-Corvair combinations, respectively, under today’s climatic conditions (Fig. 1 B). Future conditions statistically increased leaf Zn in Schlunzig-Lazio by 45% to 109 ± 8 Zn g − 1 dry leaf. Edible leaf Zn were not statistically different under future conditions in Großschirma-Butterflay, Tübingen-Metador, Schladebach-Corvair combinations compared to today's climate and were 139 ± 21, 67 ± 3 and 53 ± 3 µg Zn g − 1 dry leaf, respectively. Root Zn was 0.7-times lower than in leaves for Großschirma-Butterflay under today's climatic conditions, and 2.0-2.8-times higher for the other three soil-spinach combinations. Under future conditions, root Zn increased by 10–60%, with a significant increase for the Großschirma-Butterflay combination (Table S1A). There was a positive correlation between leaf Cd and Zn concentration in all soil-spinach combinations under both climatic conditions (Figure S2). Concentrations of two other micronutrients, Mn and Mg, increased in leaves and roots under future climatic conditions, sometimes significantly (Table S1A,B). Dry leaf yields increased significantly for Schlunzig-Lazio by 36%, Großschirma-Butterflay by 56%, Tübingen-Metador by 192% and not significantly for Schladebach-Corvair by 16% under future climatic conditions (Figure S3A). Future climatic conditions significantly increased total Cd and Zn accumulated by spinach for Schlunzig-Lazio, Großschirma-Butterflay, Tübingen-Metador and not significantly for Schladebach-Corvair (Figure S3B,C). The Cd soil-to-root transfer factor was 6-100-times higher than the Cd root-to-shoot translocation factor, depending on the soil-spinach combination (Fig. 2 A). Under today’s conditions, the Cd transfer factors for Schlunzig-Lazio, Großschirma-Butterflay, Tübingen-Metador, Schladebach-Corvair were 6.7 ± 0.8, 6.3 ± 0.4, 8.4 ± 0.9 and 20.0 ± 4.3, respectively. Future climatic conditions increased Cd transfer factors in all combinations, by 23% for Schlunzig-Lazio (8.3 ± 1.0), 37% for Großschirma-Butterflay (8.6 ± 0.8, significant), 70% for Tübingen-Metador (14.3 ± 4.2), and 56% Schladebach-Corvair (31.2 ± 8.1). The Cd root-to-shoot translocation factors under today’s climatic conditions were 0.2 ± 0.0 for Schlunzig-Lazio, Tübingen-Metador, and Schladebach-Corvair, and 1.0 ± 0.1 for Großschirma-Butterflay. Future conditions increased the translocation factor by 50% to 0.3 ± 0.0 for Schlunzig-Lazio and Tübingen-Metador. It remained 0.2 ± 0.0 for Schladebach-Corvair, but decreased by 9% to 0.9 ± 0.1 for Großschirma-Butterflay. The Cd bioconcentration factors increased in all soil-spinach combinations under future climatic conditions, with significant increases for Schlunzig-Lazio and Großschirma-Butterflay (Table S2). The Zn soil-to-root transfer was 0.35-8-times higher than the Zn root-to-shoot translocation factor, depending on the soil-spinach combination (Fig. 2 B). Under today’s conditions, the Zn transfer factors for Schlunzig-Lazio, Großschirma-Butterflay, Tübingen-Metador, Schladebach-Corvair were 0.9 ± 0.1, 0.4 ± 0.0, 2.3 ± 0.3 and 2.1 ± 0.5, respectively. Future conditions increased Zn transfer factors in all combinations, by 11% for Schlunzig-Lazio (1.0 ± 0.1), by 50% for Großschirma-Butterflay (0.6 ± 0.1, significant), by 22% for Tübingen-Metador (2.8 ± 0.7), and 57% for Schladebach-Corvair (3.3 ± 0.7). The Zn root-to-shoot translocation factors under today’s climatic conditions were 0.5 ± 0.1 for Schlunzig-Lazio, 1.4 ± 0.2 for Großschirma-Butterflay, 0.4 ± 0.0 for Tübingen-Metador, 0.5 ± 0.1 for Schladebach-Corvair. Future conditions increased the translocation by 20% to 0.6 ± 0.1 for Schlunzig-Lazio and by 50% to 0.6 ± 0.2 for Tübingen-Metador. It did not change for Großschirma-Butterflay and Schladebach-Corvair. The Zn bioconcentration factor increased in all soil-spinach combinations under future climatic conditions, except for the Tübingen-Metador combination, with a significant increase observed in the Schlunzig-Lazio combination (Table S2). The Großschirma-Butterflay combination reached the seed-producing development stage and future climatic conditions only slightly accelerated spinach development for all soil-spinach combinations (Figure S4A). Chlorophyll a and hydrogen peroxide concentrations in leaves, used here as approximation for plant health, were not affected by future climatic conditions (Figure S4B, C). Soil biogeochemical determinants of spinach metal uptake To minimize disturbance of spinach growth and root system integrity, time-series data of rhizosphere geochemical dynamics were collected only for the Großschirma-Butterflay combination. Rhizosphere exchangeable Cd, approximated with 0.01 M CaCl 2 , tended to be 21% higher under future conditions compared to today throughout the growing period, excluding the starting point (Fig. 3 A). Climate-associated differences in CaCl 2 -extractable Zn became evident from day 28 onward, with Zn tending to be higher under future conditions (Fig. 3 B). Rhizosphere pH consistently tended to be lower under future conditions compared to today, with a 0.2-unit decrease by day 55. (Fig. 3 C). Rhizosphere electrical conductivity tended to be higher under future conditions compared to today throughout the growing period reaching a 71% increase by day 55 (Fig. 3 D). At day 55, rhizosphere 16S rRNA gene and transcript copy numbers were not statistically 5.3- and 2.9-fold higher, respectively, under future conditions compared to today’s (Fig. 3 E,F). Rhizosphere soil was sampled at harvest in three soil-variety combinations not disturbed by times-series sampling. Water-extractable TC ranged from 88–155 mg g⁻¹ dry soil under today's climate, varying by combination (Fig. 3 A). Under future conditions, TC increased by 3–45%, with a significant risein Tübingen-Metador. Rhizosphere soil TOC ranged from 69–131 mg g⁻¹ under today's climate, increasing by 7% for Schlunzig-Lazio, 50% for Tübingen-Metador, and showing no change for Schladebach-Corvair. Rhizosphere soil IC ranged from 11–24 mg g⁻¹ under today's climate, rising by 12–18% across combinations under future conditions. Rhizosphere TN ranged from 13–21 mg g⁻¹ under today's climate, increasing by 6% for Schlunzig-Lazio, 5% for Tübingen-Metador, and remained unchanged for Schladebach-Corvair. Using the same extracts, 50 metabolites were measured and categorized to metabolite classes (Table S3-S7). Log2-fold-changes between climates are presented, with values above 1 indicating increased metabolite concentrations under future conditions (Fig. 3 B). Responses varied by combination, except for metal chelators, which consistently increased: 1.85 ± 0.92-log2-fold in Schlunzig-Lazio, 2.36 ± 0.41-log2-fold in Tübingen-Metador, and 3.75 ± 1.51-log2-fold in Schladebach-Corvair. Other responses were combination-specific: amino acids rose by 0.49 ± 0.37-log2-fold in Schlunzig-Lazio, fermentates by 0.69 ± 0.30-log2-fold and pathogen suppressors by 0.65 ± 0.24-log2-fold in Tübingen-Metador, and Krebs cycle intermediates by 0.40 ± 0.37-log2-fold in Schladebach-Corvair. Root-associated microbiome soil was sampled at harvest in three soil-variety combinations not disturbed by time-series sampling. Bacterial 16S rRNA gene copy numbers, alpha diversity, richness, and evenness varied by soil-spinach combination. Future climatic conditions tended to increase these metrics for Schlunzig-Lazio and Tübingen-Metador but decreased them for Schladebach-Corvair (Figure S5A,B). Root-associated bacterial community structure clustered primarily by soil-spinach combination (Fig. 5 A), explaining 60% of the variation (PERMANOVA, p < 0.001). Climatic conditions caused distinct shifts in community structure, accounting for 10% of the variation (p = 0.002), while their interaction with soil-spinach combinations an additional 8% (p = 0.04). Within the root-associated bacterial community, twelve OTUs were statistically associated with climate and at least one metal (Cd or Zn) concentration in spinach leaves, using a threshold of α = 0.05 (Fig. 5 B). Eight OTUs were identified in the Schlunzig-Lazio combination, all correlated with both Cd and Zn leaf concentrations. Of these, five OTUs ( Dokdonella, Terrimonas, Chitinophagaceae, Georgfuchsia, Ellin6055 ) increased in relative abundance, while three OTUs ( A4b, Sphingopyxis, Nannocystis ) decreased. The Schladebach-Corvair combination was the second most affected, with three OTUs ( Acidibacter, Fibrobacteraceae, Sandaracinus ) increasing in relative abundance under future conditions. All three correlated with leaf Cd concentration, with Fibrobacteraceae also correlating with Zn. Lastly, in the Tübingen-Metador combination, only one OTU ( Flavobacterium ) decreased in relative abundance under future conditions and was solely correlated with Cd concentrations in spinach leaves. DISCUSSION Future climate conditions in this study were compared to today's baseline and included a 3.4°C temperature rise, a 290 ppm v atmospheric CO₂ increase, and a 2-percentage-point reduction in soil moistureࣧthe most likely scenario for the year 2100 relative to today 20 . These conditions fostered favourable growth for spinach, leading to yields increases of 16–192%, depending on the soil-spinach combination, compared to today’s climate (Figure S3A). Elevated atmospheric CO 2 and temperature up to the plant's optimum, combined with adequate water availability, enhance photosynthesis and biomass production 14 . However, further temperature increases, reduced soil moisture, or the use of spinach varieties with different environmental optima may reduce yields 15 , 16 . Climate change impacts spinach beyond yields, affecting edible product quality, including nutrient and toxin levels. Previous reports raised concerns that CO 2 -driven plant biomass increases could dilute micronutrient concentrations in yields due to limited soil availability and tight nutrient uptake controls 21 . However, most studies focused on elevated CO 2 alone. Recent experiments combining temperature and CO 2 increases reveal a more complex relationship. Some studies suggest higher temperatures counteract CO 2 -induced micronutrient dilution in crops like soybean, wheat, and rice, likely through transpiration-driven increases in nutrient mass flow 22 , 23 . To our knowledge, this is the first study to explore these effects in leafy vegetables. Our finding supports that temperature helps maintain, or in some soil-spinach combinations, even increases concentrations of essential nutrients Zn, Mn, and Mg, under future climates (Fig. 1 ; Table S1). In contrast, harmful Cd accumulation increased by 25–50% under future conditions across all combinations (Fig. 1 ). This greenhouse study provides the first evidence that future climatic conditions enhance metal transfer from oxic soils to crops. Climate-induced Cd accumulation in spinach leaves may result from changes in its soil mobility, soil-to-root transfer, and root-to-shoot translocation, discussed in the following sections. For Cd uptake in spinach, it must first be converted into phytoavailable forms, such as dissolved ions or weakly adsorbed species on soil particles. Climate-driven increases in metal mobility have been shown in other systems, such as arsenic in rice rhizospheres, where redox processes, particularly microbially mediated reductive dissolution of iron and arsenic, enhance mobility in flooded, anoxic paddy soils 12 . However, these redox processes are less relevant for oxic soils and non-redox-active Cd. A recent study by Drabesch et al. using similar climatic variables to those applied here demonstrated that future climatic conditions influence Cd dynamics in oxic soils by altering soil microbiomes 10 . Specifically, ammonium oxidation stimulated by climate-induced shifts lowered soil pH, displacing Cd through proton activity, in Drabesch et al. observed in Tübingen soil 10 , here confirmed in Großschirma soil. Acidification dissolves Cd co-precipitates, increases soil particle surface charge, and reduces Cd binding via electrostatic repulsion 24 . In support of Drabesch et al 10 , we also found taxa-based evidence for enhanced N cycling in the Schlunzig-Lazio combination, the soil with the highest total Cd content. Two root-associated taxa ( Dokdonella, Chitinophagaceae ; Table S6) statistically correlated with both climate and Cd were identified as potential nitrifiers, and showed increased abundance under future conditions (Fig. 5 B). Increased N pools, particularly amino acids in the rhizosphere (Fig. 4 B), may stem from plant defence mechanisms against Cd 25 , circumstantially providing substrates for ammonium oxidation and further mobilizing Cd. Unlike Drabesch et al., who excluded plants in their study and observed no differences in dissolved carbon 10 , we found a tendency for climate-induced increases in rhizosphere water-extractable carbon (Fig. 4 A). This is likely driven by enhanced photosynthesis-derived carbon transfer to the soil via root exudation 26 . Spinach and soil microbiomes exhibited increased growth and activity under future climatic conditions (Figure S3; Fig. 3 ), elevating nutrient demands. Both likely excreted metabolites to mobilize nutrients, including chelators, whose collective concentrations significantly increased across three soil-spinach combinations (Fig. 4 B). However, chelators may inadvertently mobilize Cd and not just nutrients 27 , contributing to Cd’s increased soil mobility. This mechanism was crucial even in high-pH soils, for example, Cd accumulation in the Schladebach-Corvair combination occurred despite a soil pH of 7.6 (Figure S1). Moreover, while Drabesch et al. found future climatic conditions altered soil carbon composition toward larger, more oxidized, lower energy organic matter 10 , our inclusion of plant inputs maintained fresh carbon sources (Fig. 4 B), fostering favorable conditions for bacterial growth. Soil- and root-associated bacterial abundance (Fig. 1 E; Figure S5A) and activity increased, evidenced by higher 16S rRNA transcript numbers (Fig. 1 F) and elevated Krebs cycle metabolites in the soil (Fig. 4 B). In the Tübingen-Metador combination, elevated fermentative metabolites suggested organic matter turnover was so intense that anoxic microenvironments may have formed 28 . Enhanced bacterial metabolism may have increased soil carbon turnover and decomposition, raising electrical conductivity (Fig. 3 D) and further mobilizing Cd. Organic acids from carbon sources, Krebs cycle products, and fermentative metabolites (Table S3-S7) lowered soil pH (Fig. 3 C) with impacts on Cd mobility as discussed above. Soil Cd mobility increased earlier and more significantly than Zn during the growth period (Fig. 3 A,B), resulting in higher Cd than Zn accumulation in spinach leaves (Fig. 1 A,B). This aligns with previous findings, as Zn’s lower first hydrolysis constant and larger hydrated radius increase its tendency to adsorb and precipitate in soils compared to Cd 29 , 30 . In addition to altering metal accumulation in spinach, climatic condition and potentially climate-enhanced soil metal mobility shifted root-associated microbiome composition, potentially affecting long-term soil fertility, plant performance, and ecosystem functioning 31 . The Schlunzig-Lazio combination, with the highest total soil Cd and Zn (Figure S1A) and the only combination where leaf Zn increased under future conditions (Fig. 1 B), exhibited the most root-associated taxa significantly correlated with climate and phytoavailable Cd and Zn (Fig. 5 B). In this soil, climate-enhanced metal mobility may reach stress-inducing levels, evidenced by increased abundances of metal-resistant and metal-immobilizing taxa ( Georgfuchsia , Ellin6055, Terrimonas ; Table S6). Conversely, negatively affected taxa included complex organic carbon degraders (A4b, Sphingopyxis, Nannocystis ; Table S6), suggesting metal toxicity shifts the microbiome toward oligotrophs, potentially disrupting future soil nutrient cycling. The second most affected root-associated community was in the Schladebach-Corvair combination (Fig. 5 B). Despite low soil Cd and Zn (Figure S1A), increased metal mobility may not yet be toxic but stimulatory, driving microbes to seek additional energy sources for resistance 32 . This likely promoted an increase in complex organic carbon degraders and metal-resistant taxa ( Acidibacter, Fibrobacteraceae, Sandaracinus ; Table S6). Lastly, the least affected root-associated community was in the Tübingen-Metador combination (Fig. 5 B), which had the second-lowest total metal concentrations (Figure S1A) and the highest carbon and nitrogen pools (Fig. 4 A). This nutrient abundance likely supported bacterial stability, minimizing community changes. However, the only taxon significantly affected by climate-enhanced Cd mobility was the plant-growth promoter Flavobacterium (Table S6), suggesting its potential vulnerability to future Cd mobilization. Once mobilized, metal cations move towards the roots via diffusion and advection. Diffusion occurs as cations are attracted to the negatively charged root surface, creating a concentration gradient, while advection transports ions with water moving toward roots due to plant transpiration 27 . Future conditions likely enhanced advective transport as elevated atmospheric CO₂ increases stomatal opening, leading to greater water evaporation from leaves, further amplified by higher temperatures 16 . Cadmium, being more mobile in soil solution than Zn (Fig. 3 A,B), likely adsorbed more readily to root surfaces, with increased transpiration boosting passive Cd uptake relative to Zn 27 . As an essential micronutrient, Zn uptake exceeds passive processes, requiring active transport mechanisms like ZIP family proteins in spinach roots 33 . However, due to its chemical similarity to Zn, Cd can also be mistakenly transported via Zn transporters 33 , likely intensified by its increased soil mobility. Under excess metal concentrations in root cell cytosol, homeostatic mechanisms regulating Zn and Cd involve efflux into compartments like the apoplast, vacuole, and Trans-Golgi Network 27 , immobilizing metals to reduce toxicity but retaining them in root tissues. Overall, future climatic conditions enhanced soil-to-root transfer for both Cd and Zn, with Cd transfer factors increasing by 46% on average compared to 36% for Zn across soil-spinach combinations (Fig. 2 A, C). Metals taken up by roots are transported to aerial parts via the xylem, driven by transpiration and mediated by metal transporters. This tightly regulated root-to-shoot translocation varies among spinach varieties, influencing final leaf Zn and Cd concentrations 34 . Zinc root-to-shoot translocation factors tended to increase under future climatic conditions in only half the tested combinations (Fig. 2 D). Despite climate-induced biomass increases, leaf Zn concentrations remained stable in most varieties, reflecting robust variety-specific Zn homeostasis. Only the Schlunzig-Lazio combination showed a climate-induced increase in leaf Zn (Fig. 1 B), possibly due to high Zn in Schlunzig soil (Figure S1A), which, when mobilized under future climate, may have exceeded Lazio's Zn immobilization capacity. If Cd is not immobilized in roots, it moves to shoots exploiting Zn transport pathways or passive diffusion along transpirational fluxes 27 , 33 . Under future conditions, Cd translocation to shoot increased more than Zn, with a 40% average rise in root-to-shoot translocation factors (Fig. 2 B). This matches the 46% rise in Cd soil-to-root transfer factors, confirming spinach efficiently transports Cd to shoots 18 and highlighting the soil-to-root barriers importance, as spinach can enhances leaf translocation without signs of toxicity (Figure S4). Research on climate change impacts on food security has traditionally focused on yield quantity. Here, we demonstrated for the first time that Cd accumulation by plants, a non-redox-active metal and major contaminant in oxic soils (90% of agricultural soils 13 ), is also affected by future climatic conditions. This was not matched by equal increases in essential nutrients like Zn, Mn, and Mg, which varied depending on the plant-soil combination. Under the experimental conditions, future climate could increase Cd accumulation in spinach, potentially raising consumer exposure to harmful Cd levels and posing financial risks to agriculture due to safety standard non-compliance. These findings likely extend to other leafy vegetables and warrant further study on staple grain crops like wheat. Our results show that future climates not only boost Cd transfer from soil to roots but also enhances its translocation to leaves. Mitigation strategies such as soil management techniques that immobilize Cd more effectively than Zn (e.g. organic amendments 35 ) and breeding low-Cd spinach varieties 36 could help address these risks. MATERIAL AND METHODS Soil and spinach characterization. To ensure that the findings in this study are applicable to a wide range of soils and spinach variety, four unique combinations of soil types and spinach varieties were selected for this study (Figure S1). Four agricultural soils, originating from distinct geographic locations and therefore of different geochemical properties, were used. They are denoted based on their geographic origins within Germany as following: Schlunzig (50°47'29.3"N 12°30'02.6"E), Großschirma (50°57'42.7"N 13°16'44.6"E), Tübingen (48°32'48.0"N 9°02'30.7"E), and Schladebach (51°18'30.6"N 12°06'16.4"E). Soils were consistently collected from the top 20 cm, mixed well, air-dried, sieved through a 4 mm mesh sieve, and stored in the dark. The time of sampling and geochemical characteristics of each soil are summarized in Table S7. Unless otherwise stated in the cited sources, the following protocols were used for soil characterization. Texture was determined in triplicates by a hydrometer in sodium hexametaphosphate solution. The fraction of sand was quantified with a hydrometer after 40 s, silt after 2 h, and the remaining fraction was calculated as clay. Soil pH was quantified in triplicates from air-dried soil with 0.01 M CaCl 2 at a 1:5 w/v ratio after 24 h at room temperature. The cation exchange capacity was quantified in triplicates from air-dried soils with 0.1 M BaCl 2 (analytical grade) at a 1:25 w/v ratio for 4 h of shaking (200 rpm) at room temperature. The total carbon and nitrogen contents were quantified in triplicates from 40°C-dried soils by combustion in tin foil balls (Flash 2000 Organic Elemental Analyzer, Delta V Advantage Isotope Ratio MS). The elemental contents of the soils were quantified in 10 mm thick soil powders dried at 105°C using X-ray fluorescence (XRF, SPECTRO XEPOS spectrometer, SPECTRO Analytical Instruments GmbH, Germany). To maximize Cd quantification, region 2 between 6 and 15 keV was extended to 900 ms. Four spinach varieties commonly used in Europe were selected for this study: Lazio, Butterflay, Metador, Corvair (Table S7). The seeds were stored in the dark at room temperature until the experiment. Greenhouse setup and pot study design. The Großschirma soil – Butterflay spinach combination was grown in Spring 2023 and the other three soil-spinach combinations in Autumn 2023 in daylight-fed greenhouses at the UFZ Research Station in Bad Lauchstädt (51°23'33.6"N 11°52'33.6"E). For today’s climate variables, we examined midday temperatures of 20°C and ambient atmospheric CO 2 , which represent commonly used for spring and fall cultivation of the used spinach varieties according to suppliers’ recommendation (Table S8). Future climatic conditions applied in this study are compared to today's baseline and included a 3.4°C temperature increase, a 290 ppm v rise in atmospheric CO₂. These conditions align with the projected climate scenario under Shared Socioeconomic Pathway SSP2–4.5 19 , identified as the most likely scenario for the year 2100 relative to today 20 , and a SSP4-8.5 scenario relative to pre-industrial times. To ensure chamber-independent results, three different chambers were used and their climatic conditions were rotated between experimental runs. Black plastic pots (9x9x20 cm) were filled with ~ 1.4 kg of air-dried soil. The first irrigation was carried out with tap water by capillarity overnight. After reaching saturation, four spinach seeds that had been soaked for two days (Großschirma- Butterflay combination) or one day (all other soil-spinach combinations) in autoclaved Milli-Q® water were planted in pots 1 cm deep and 1.5 cm apart. Young seedlings were thinned to 2 plants (Großschirma- Butterflay combination) or 1 plant (all other soil-spinach combinations) within the first 2 weeks. Irrigation, conducted twice a week, remained consistent across both climate conditions and resulted in an average of 47% WHC under today's climatic conditions. However, due to higher atmospheric temperatures in the future climate scenario, soil moisture levels were lower, averaging 45% WHC (Figure S6). Each soil-spinach variety combination in each climate was carried out with twelve replicates using a randomized block design 37 . To avoid biases in light exposure, atmospheric temperature, and drafts in the growth chambers, the blocks were moved weekly. Time-resolved soil geochemical data were obtained for five to seven of the twelve replicates of one of the Großschirma-Butterflay soil-variety combination to minimize disturbance to spinach growth and maintain roots systems integrity in the other varieties and replicates. Soil samples were collected to monitor CaCl 2 -extractable Cd and Zn, pH, and electrical conductivity. Five-cm deep soil cores were taken on days 1, 8, 29 and 55, randomly rotated among replicates of the Großschirma-Butterflay combination so that each replicate was sampled twice during the experiment. Cores were thoroughly mixed, aliquoted, flash-frozen in dry ice, and stored at -20°C until analyses. At harvest, rhizosphere soil samples for bacterial analysis were collected from three replicates using the same method and stored at -80°C for subsequent analyses (see bacterial analysis section). For rhizosphere soil solution carbon profiling, soil samples were collected at harvest from the three remaining soil-variety combinations that had not been disturbed by time-series sampling. Horizontal cores were taken at a depth of 10 cm to obtain rhizosphere soil at harvest. These cores were thoroughly mixed, roots removed, aliquoted, flash-frozen in dry ice, and stored at -20°C for water-extractable root exudate, carbon and nitrogen analysis (see soil analysis section). Plant-related analyses were done for all replicates. Plant growth and development were tracked weekly over the duration of cultivation. At time of harvest, spinach plants were first photographed (Figure S1B). To minimize photosynthesis-related artifacts in plant and soil data, sampling was organized in blocks, and done in parallel per climate. Edible yields were quantified by wet and dry mass, washed in deionised water, and flash-frozen in dry ice until storage at -80°C before analysis of metal contents and plant health (see plant analysis section). Roots were separated from soil, washed thoroughly in deionised water, weighed and flash-frozen in dry ice until storage at -80°C before analysis of metal contents (all replicates) and root-associated bacteria (three replicates from the three soil-variety combinations that were not disturbed by time-series soil sampling). Plant analysis. Edible yields and root mass were normalized to their dry weight (40°C-dried until constant weight). For metal and plant health quantification, spinach was ground in liquid nitrogen. Metal contents of edible yields and roots were assessed from 12 biological replicates by extracting approximately 0.1 g biomass in 3 mL of 65% nitric acid (HNO 3 , analytical grade) and 2 mL of 30% H 2 O 2 (analytical grade) in a microwave digester (Mars6, CEM, USA) with a 15-min ramp phase to 15 min at 180°C, followed by a 30 min cool-down phase 12 . Extractants were filtered through filter paper (Whatman qualitative filter paper, Grade 1) and diluted in MQ water. Extraction blank and a reference material (European Reference Material ERM®-CD281) were extracted along with each extraction run for later computational comparison and alinement between runs. Elemental contents were quantified in leaf extraction filtrates with XXX. Leaf chlorophyll a was quantified by extracting ground, frozen leaves with 80% acetone and measuring absorbances spectrophotometrically at 663 and 645 nm. To evaluate climate stress in spinach, H₂O₂ contents were determined from 0.1% (w/v) trichloroacetic acid (TCA) extracts of frozen, ground leaf tissues at a 1:7.5 w/v ratio. Subsequently, 300 µL of 10 mM potassium phosphate buffer (pH 7) and 600 µL of 1 M potassium iodide were added to 300 µL of the 0.1% TCA extract. The mixture was incubated in the dark for 20 minutes, and absorbance was measured at 390 nm. H₂O₂ content was calculated using a standard curve. Soil analysis. Mobile Cd and Zn in soil were approximated using six biological replicates by a 15 minute extraction with 0.01 M CaCl 2 at a 1:6.67 w/v ratio at room temperature 38 . Extractants were filtered through 0.22 µm filters (Minisart® high flow, Sartorius, Germany), which were pre-washed with MQ water (to remove filter-associated organic matter impurities). Afterward, they were acidified with HNO 3 to a concentration of 2% and stored at 4°C until quantification by ICP-MS and ICP-OES as described above for plant material. Electrical conductivity and soil pH were measured using five-seven replicates at a fresh soil to ultra-pure water ratio of 1:5 (w/v). Electrical conductivity was determined after in the settling solution, and soil pH after 24 hours (SD335 Multi-Parameter Meter, Lovibond, Germany). Carbon and nitrogen profiles were extracted using 4 biological replicates by a 4 hour extraction with ultra-pure water at a 1:8 w/v ratio at room temperature 39 . Extractants were filtered as described for CaCl 2 extraction and stored at -20°C until analysis for total carbon (TC), total organic carbon (TOC), inorganic carbon (IC) and total nitrogen (TN) concentrations with a TOC analyzer (Multi N/C 2100S duo, Analytik Jena, Germany). The concentration of metabolites was measured using GC-MS (Shimadzu GC/MS TQ 8040, Japan) operated in Electron Ionisation mode (EI). Metabolites were measured either by headspace injection (MS acquisition mode: Scan) or as a liquid sample after derivatization (MS acquisition mode: MRM). For derivatization, samples were thawed on ice and 3000 pmol 13C-glucose was added as an internal standard to 250 µL sample prior to freeze-drying. The solids were re-dissolved with 50 µL methoxamine (20 mg mL − 1 pyridine), ultrasonicated for 10 min and subsequently incubated at 30° C for 90 min. Next, 70 µL N-methyl-N-(trimethylsilyl)-trifluor-acetamid (MSTFA) was added and samples were incubated at 40°C for 60 min, after which they were kept at room temperature for 2 h{Fiehn, 2000 #294}. A volume of 1 µL was used for injection in splitted mode on a Restek SH-Rxi-5SIL MS column (30 m; film 0.25 µm; diameter 0.25 mm). The injection port was heated to 280°C. For chromatographic separation a He flow rate of 1.1 mL min-1 and a temperature increase of 10°C min-1 from 100 to 320°C were employed. For the measurement via the headspace method, 250 µL of thawed sample together with 2500 pmol octanol as internal standard were added into 10 mL glass vials with closed lids. The total liquid amount is 1 mL containing 470 µL saltout media (882 g L − 1 Ammonium sulfate and 238 g L − 1 Sodium dihydrogen phosphate, 20 µL Phosphoric acid 85% and adjusted with MiliQ water to the end volume{Fiorini, 2016 #296}. After heating and shaking at 80°C for 30 min a volume of 1 mL of headspace gas was injected onto the column Stabilwax-DA (30 m; film 1 µm; diameter 0.32 mm) from Restek. The He flow rate was 1.1 mL min-1 and the column temperature increased by 10°C min − 1 from 40 to 240°C{Zhang, 2018 #295}. Concentrations were quantified with a 8-point external calibration containing standards of all targeted analytes, ranging from 20 to max. 1.00E + 07 pmol per 250 µL sample for headspace method and 20 to max. 3.00E + 04 pmol per 250 µL sample for liquide sample method. Labsolutions Insight (Shimadzu, Japan) GCMS software was used for peak integration which was double checked manually. Relative peak areas and amount of metabolites were calculated with R using a self programmed shinyapp in Rstudio{Chang, 2025 #297}. Final metabolite data were annotated by class (Table S3-S5), with log2 fold changes between climate scenarios calculated for each class and block, followed by averaging these changes for each class and determining errors through propagation. All soil analyses were normalized to dry weight (105°C-dried until constant weight). Bacterial analysis. To analyze bacterial quantity and taxonomic diversity, nucleic acids were extracted following Lueders et al., 2004 40 , using 0.6 g of wet soil. The quality and quantity of DNA and RNA were verified by NanoDrop 2000c (ThermoFisher, USA) and fluorometric quantification with Invitrogen™ Qubit™ 3 Fluorometer Qubit® 3.0 Fluorometer (ThermoFisher, USA), respectively. Copy numbers of the rhizosphere soil and root-associated bacterial 16S rRNA gene and transcript were amplified and quantified by qPCR on a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Germany). As a standard, the plasmid vector pCR2.1® (Invitrogen, Darmstadt, Germany) containing a cloned 16S rRNA gene fragment of Thiomonas sp. was used 41 . A master mix was prepared with 1× SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, United States), 75 nM of primer 341-F (5′-CCTACGGAGGCAGCAG-3′) 42 , and 225 nM of primer 797-R (5′- GGACTACCAGGGTATCTAATCCTGTT-3′) 43 . In a total volume of 10 µl, 1 µl DNA extract (500-fold diluted) or standard plasmid DNA (eight-fold dilution series) and 9 µl master mix were quantified in 96-well PCR plates (Bio-Rad Laboratories). The qPCR program was run with 3 min at 98°C, followed by 40 cycles of 15 s at 98°C and 30 s at 60°C. For verification, melting curve analysis was performed using CFX Manager™ software. Data were obtained from biological triplicates, except for the Großschirma-Butterflay combination, where one sample was lost, leaving only two replicates for analysis. Results were normalized to the dry weight of 105°C-dried soil. Root-associated bacterial 16S rRNA genes and transcripts were amplicon-sequenced using primers 515F and 806R 44 . The quality and quantity of the purified amplicons were assessed via agarose gel electrophoresis. Library preparation (Nextera, Illumina, USA) and sequencing were carried out using the 2×250 bp MiSeq Reagent Kit v2 on an Illumina MiSeq sequencing system (Illumina, San Diego, USA). Bacterial raw reads quality-checked sequences and bioinformatics assembly of datasets were performed using a workflow primarily based on DADA2{Callahan, 2016 #298}. This was implemented through the standardized pipeline Dadasnake version 0.11{Weißbecker, 2020 #299}. Primers were detected with a maximum allowance of 20% mismatch and were trimmed using Cutadapt version 1.18. Bacterial sequences were filtered with the following parameters: trunc_quality:13, trunc_length:170 bp for both forward and reverse and max_EE:0.2. Taxonomic assignment was conducted using the SILVA database v138.1{Quast, 2012 #162}. ASVs were clustered using VSEARCH with a threshold of 97%, resulting in operational taxonomic units (OTUs). To identify root-associated OTUs related to climate and Cd/Zn concentrations in spinach leaves, the following pipeline was applied: first, Spearman correlations were conducted to identify OTUs correlated with Cd or Zn concentrations. These OTUs were then tested using a t-test between current and future climatic conditions to determine those significantly affected. Data analyses, statistical evaluation and data visualization. We utilized the R programming language and conducted our analyses within the RStudio integrated development environment for statistical computing and graphics. Statistical analyses focused on comparing today versus future conditions within each soil-spinach combination, as comparing different soil-spinach combinations was not the goal of this study. T-tests and Welch’s tests (for unequal variances) were used for pairwise comparisons, while a linear mixed model with repeated measures assessed climate impacts on time-series data, with Tukey’s HSD (α = 0.05) for post-hoc adjustments. Pearson correlation analyzed Cd and Zn concentrations in leaves within each soil-spinach combination. PERMANOVA assessed climate effects on root-associated bacterial community composition across all soils, as replicates were insufficient for analysis within individual soil-spinach combinations. Three factors were calculated to estimate Zn and Cd mobilization within the soil-plant system. The bioconcentration factor (1) was calculated to estimate the transfer of metals from the soil into the total biomass of spinach plants. $$\:\text{B}\text{i}\text{o}\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{f}\text{a}\text{c}\text{t}\text{o}\text{r}=\frac{\text{S}\text{h}\text{o}\text{o}\text{t}+\text{R}\text{o}\text{o}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{s}\text{o}\text{i}\text{l}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}$$ 1 The soil-to-root translocation factor (2) was calculated to estimate the transfer of metals from the soil specifically to the roots of spinach plants. $$\:\text{S}\text{o}\text{i}\text{l}\:\text{t}\text{o}\:\text{r}\text{o}\text{o}\text{t}\:\text{t}\text{r}\text{a}\text{n}\text{s}\text{f}\text{e}\text{r}\:\text{f}\text{a}\text{c}\text{t}\text{o}\text{r}=\frac{\text{R}\text{o}\text{o}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{s}\text{o}\text{i}\text{l}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}$$ 2 The root-to-shoot translocation factor (3) was calculated to assess the internal transfer of metals from the roots to the shoots within spinach plants $$\:\text{R}\text{o}\text{o}\text{t}\:\text{t}\text{o}\:\text{s}\text{h}\text{o}\text{o}\text{t}\:\text{t}\text{r}\text{a}\text{n}\text{s}\text{l}\text{o}\text{c}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{f}\text{a}\text{c}\text{t}\text{o}\text{r}=\frac{\text{S}\text{h}\text{o}\text{o}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{R}\text{o}\text{o}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}$$ 3 Declarations COMPETING INTERESTS: The authors have no competing interests, financial or non-financial, in relation to the work described within this manuscript. AUTHOR CONTRIBUTION This study was conceptualized by AP, AG and EMM. Experiments were planned by AP, AG with input from EMM, and plant growth support by IM. The experiments, plant maintenance, lab work and analysis were carried out by AP, AG, SK, PGR, NS, LDPS, EMM. Elemental measurements were carried out by MH. Metabolite measurements were carried out by JK and MS. Data interpretation was done by AP with primary input from EMM, TR, LDPS, MH and JK. The manuscript and supporting information were written by AP and EMM with additional input from all co-authors. Funding for the project was acquired by EMM. ACKNOWLEDGEMENTS: We thank Marie Mollenkopf, Carolina Vergara Cid, Karolin Seiferth, Hendrik Seifert, Nawshin Atia, Paula Gscheidel, Jennifer Horstmann, Aaron Jakob, Lieke Lipsch, Ayushi Parmar, Swati Sharma, Birgit Sawall for lab and greenhouse support. The microbial community composition data have been computed at the high-performance computing cluster EVE, a joint effort of both the Helmholtz-Center for Environmental Research-UFZ and the German Center for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig. We kindly thank the server admins. The Saxonian State Agency for Environment, Agriculture and Geology, especially Dorit Julich and Ingo Müller, and Eckart Wizemann for supply of soil. This work was financed through the Helmholtz Young Investigator Grant RhizoThreats. DATA AVAILABILITY STATEMENT: The datasets generated during and/or analysed during the current study are also available from the corresponding authors on reasonable request. References Pinstrup-Andersen P (2009) Food security: definition and measurement. Food Secur 1:5–7 Zoroddu MA et al (2019) The essential metals for humans: a brief overview. 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Water Sci Technol 32:1–9 Gensberger ET et al (2014) Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality. Water Res 67:367–376 Caporaso JG et al (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences 108, 4516–4522 Additional Declarations The authors declare no competing interests. Supplementary Files ClimateMetalSpinachPienkowskaetalSupplement.docx Supplement_Pienkowska_et_al Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5947512","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":410217054,"identity":"29a34b78-771c-4b73-8963-7bed901bea66","order_by":0,"name":"Aleksandra Pieńkowska","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Pieńkowska","suffix":""},{"id":410217055,"identity":"2fe00757-9b7a-4614-8a6c-b35dc0f79cd6","order_by":1,"name":"Alexandra Glöckle","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Glöckle","suffix":""},{"id":410217056,"identity":"acad392a-0147-452a-900d-fe3c5e7b4a83","order_by":2,"name":"Natalia Sánchez","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Sánchez","suffix":""},{"id":410217057,"identity":"2f824a54-139d-437b-81d5-a7f82b046e12","order_by":3,"name":"Shitalben Khadela","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Shitalben","middleName":"","lastName":"Khadela","suffix":""},{"id":410217058,"identity":"f0b71843-c0c2-481b-8eb0-4f8eaf17c25d","order_by":4,"name":"Paul-Georg Richter","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Paul-Georg","middleName":"","lastName":"Richter","suffix":""},{"id":410217059,"identity":"61c0d73d-c99f-48ce-9884-b41c082172b3","order_by":5,"name":"Ines Merbach","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Ines","middleName":"","lastName":"Merbach","suffix":""},{"id":410217060,"identity":"1a41a8d4-d43e-4a2f-9544-1e91c0947c28","order_by":6,"name":"Martin Herzberg","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Herzberg","suffix":""},{"id":410217061,"identity":"22f4481c-04d2-4bc6-a17f-e9d27c0cc36d","order_by":7,"name":"Joachim Kilian","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Kilian","suffix":""},{"id":410217062,"identity":"7eeb5c2f-1e3c-425a-b04a-f3621de54732","order_by":8,"name":"Mark Stahl","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Stahl","suffix":""},{"id":410217063,"identity":"6e5894c3-f3c7-438b-9457-47d77427a55e","order_by":9,"name":"Luis Daniel Prada Salcedo","email":"","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Daniel Prada","lastName":"Salcedo","suffix":""},{"id":410217064,"identity":"eb82ec3a-a8e7-4aa1-8aa9-80391bbbd51d","order_by":10,"name":"Thomas Reitz","email":"","orcid":"","institution":"Martin-Luther-Universität Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Reitz","suffix":""},{"id":410217065,"identity":"39d17ade-f1d8-4b6a-bc21-b291b05539c0","order_by":11,"name":"E. Marie Muehe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYJCCA2BSgoHxAYMNnEucFmYDhjQitTBAtbBJEKVFt/3swwMfGLYl9ks3H6vmSahj4DvegF+L2Zl0g4MzGG4nzpxzLO02TwIbg+QZAtaYHUhjOMwD1LLhRo7Zbd4fPAwGNxIIaDn/jOHwH6CW/TfyvxXzJEgwGNx/QEDLDaAtDCBbJHLYmHkSDIC24NcB1PKM4WCPwW3jGTfSjCXnJCTwSJ4h6LA05g8/Km7L9s9IfvjhTUKdHN/xAwSsAQMDBJOHGPWjYBSMglEwCggAAPj4SMMsyn4MAAAAAElFTkSuQmCC","orcid":"","institution":"Helmholtz Centre for Environmental Research UFZ","correspondingAuthor":true,"prefix":"","firstName":"E.","middleName":"Marie","lastName":"Muehe","suffix":""}],"badges":[],"createdAt":"2025-02-02 22:03:53","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5947512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5947512/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75395474,"identity":"03a4c12d-c6dc-4641-9440-4e3c574bca87","added_by":"auto","created_at":"2025-02-04 06:27:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166738,"visible":true,"origin":"","legend":"\u003cp\u003eMetal concentrations in edible yields of spinach grown under different climatic conditions. (\u003cstrong\u003eA\u003c/strong\u003e)\u0026nbsp;Leaf cadmium and (\u003cstrong\u003eB\u003c/strong\u003e)\u0026nbsp;zink concentrations normalized to dry weight of four soil-spinach combinations: Schlunzig soil with Spinach var. Lazio (red, Cd\u003csub\u003esoil\u003c/sub\u003e = 1.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =192ppm\u003csub\u003ew\u003c/sub\u003e), Großschirma soil with Spinach var. Butterflay (yellow, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.6 ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =188 ppm\u003csub\u003ew\u003c/sub\u003e), Tübingen soil with Spinach var. Metador (green, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.2 ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =77 ppm\u003csub\u003ew\u003c/sub\u003e), Schladebach soil with Spinach var. Corvair (blue, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =34ppm\u003csub\u003ew\u003c/sub\u003e) under today’s (light color, mid-day 20.5°C, 500 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 47 % WHC\u003csub\u003emax\u003c/sub\u003e) and future (dark color, mid-day 23.9°C,\u0026nbsp; 800 ppmv CO\u003csub\u003e2\u003c/sub\u003e, 45 % WHC\u003csub\u003emax\u003c/sub\u003e) climatic conditions. Twelve biological replicates were compared for climatic impact within each soil-spinach combination using t-tests. The p-values are represented as follows: \"***\" = p \u0026lt; 0.001, \"**\" = p \u0026lt; 0.01, \"*\" = p \u0026lt; 0.05, and \"NS\" = p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/767ca58a97d072600e71316e.png"},{"id":75395473,"identity":"0d5429ec-65ec-4409-ab41-2b0246a5e570","added_by":"auto","created_at":"2025-02-04 06:27:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126511,"visible":true,"origin":"","legend":"\u003cp\u003eMetal soil-to-root transfer and root-to-shoot translocation factors of spinach grown under different climatic conditions. (A) cadmium and (B) zink factors of four soil-spinach combinations: Schlunzig soil with Spinach var. Lazio (red, Cd\u003csub\u003esoil\u003c/sub\u003e = 1.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =192ppm\u003csub\u003ew\u003c/sub\u003e), Großschirma soil with Spinach var. Butterflay (yellow, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.6 ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =188 ppm\u003csub\u003ew\u003c/sub\u003e), Tübingen soil with Spinach var. Metador (green, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.2 ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =77 ppm\u003csub\u003ew\u003c/sub\u003e), Schladebach soil with Spinach var. Corvair (blue, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =34ppm\u003csub\u003ew\u003c/sub\u003e) under today’s (light color, mid-day 20.5°C, 500 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 47 % WHC\u003csub\u003emax\u003c/sub\u003e) and future (dark color, mid-day 23.9°C, \u0026nbsp;800 ppmv CO\u003csub\u003e2\u003c/sub\u003e, 45 % WHC\u003csub\u003emax\u003c/sub\u003e) climatic conditions. Twelve biological replicates were compared for climatic impact within each soil-spinach combination using t-test. The p-values are represented as follows: \"***\" = p \u0026lt; 0.001, \"**\" = p \u0026lt; 0.01, \"*\" = p \u0026lt; 0.05, and \"NS\" = p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/d6095e7dce8946f30a0335c6.png"},{"id":75395476,"identity":"71c8df5f-d644-4d7e-b79e-3ee3a89359c0","added_by":"auto","created_at":"2025-02-04 06:27:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":268101,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Mobile cadmium, (B) mobile zinc, (C) pH, (D) electrical conductivity, (E) bacterial 16S rRNA gene copy number at harvest, and (F) bacterial 16S rRNA transcript copy number at harvest of Großschirma rhizosphere soil with Spinach var. Butterflay (Cd\u003csub\u003esoil\u003c/sub\u003e = 0.6 ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =188 ppm\u003csub\u003ew\u003c/sub\u003e) under today’s (light colored empty symbols, mid-day 20.5°C, 500 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 47 % WHC\u003csub\u003emax\u003c/sub\u003e) and future (dark colored filled symbols, mid-day 23.9°C, 800 ppmv CO\u003csub\u003e2\u003c/sub\u003e, 45 % WHC\u003csub\u003emax\u003c/sub\u003e) climatic conditions. (A-D) Five-seven biological replicates; mean values ± standard errors (excluding starting samples) were compared using a linear mixed model with repeated measures and Tukey's HSD (α = 0.05). (C-D) two-three biological replicates; mean values ± standard errors were compared for climatic impact within each soil-spinach combination using Welch's t-test for unequal variance. Due to the low number of replicates, these statistical results should be interpreted with caution. The p-values are represented as follows: \"***\" = p \u0026lt; 0.001, \"**\" = p \u0026lt; 0.01, \"*\" = p \u0026lt; 0.05, and \"NS\" = p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/8ecc071366153762bb16ae69.png"},{"id":75395486,"identity":"5f170bac-c08b-470e-9214-826acb6d653d","added_by":"auto","created_at":"2025-02-04 06:27:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297169,"visible":true,"origin":"","legend":"\u003cp\u003eCarbon and nitrogen profiles of spinach rhizosphere soil under different climatic conditions. (A) Water-extractable total carbon, total organic carbon, total inorganic carbon, and total nitrogen. (B) Log2 fold-change in water-extractable metabolite concentrations between today and future climate scenarios of three soil-spinach combinations: Schlunzig soil with Spinach var. Lazio (red, Cd\u003csub\u003esoil\u003c/sub\u003e = 1.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =192ppm\u003csub\u003ew\u003c/sub\u003e), Tübingen soil with Spinach var. Metador (green, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.2ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =77ppm\u003csub\u003ew\u003c/sub\u003e), Schladebach soil with Spinach var. Corvair (blue, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =34ppm\u003csub\u003ew\u003c/sub\u003e) under today’s (light color, mid-day 20.5°C, 500 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 47 % WHC\u003csub\u003emax\u003c/sub\u003e) and future (dark color, mid-day 23.9°C, \u0026nbsp;800 ppmv CO\u003csub\u003e2\u003c/sub\u003e, 45 % WHC\u003csub\u003emax\u003c/sub\u003e) climatic conditions. (A) Four biological replicates, mean values ± standard errors were compared for climatic impact within each soil-spinach combination using t-test. The p-values are represented as follows: \"***\" = p \u0026lt; 0.001, \"**\" = p \u0026lt; 0.01, \"*\" = p \u0026lt; 0.05, and \"NS\" = p \u0026gt; 0.05. (B) Four biological replicates, mean values ± error propagation.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/d643eaca69b77811a8aad061.png"},{"id":75395682,"identity":"480bd5d1-9cf1-48b4-9e80-a3c5d1bdd54d","added_by":"auto","created_at":"2025-02-04 06:35:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":370827,"visible":true,"origin":"","legend":"\u003cp\u003eRoot-associated bacterial community structure. (A) NMDS of microbiome at harvest (day 63) of three soil-spinach combinations: Schlunzig soil with Spinach var. Lazio (red, Cd\u003csub\u003esoil\u003c/sub\u003e = 1.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =192ppm\u003csub\u003ew\u003c/sub\u003e), Tübingen soil with Spinach var. Metador (green, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.2ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =77ppm\u003csub\u003ew\u003c/sub\u003e), Schladebach soil with Spinach var. Corvair (blue, Cd\u003csub\u003esoil\u003c/sub\u003e = 0.1ppm\u003csub\u003ew\u003c/sub\u003e, Zn\u003csub\u003esoil\u003c/sub\u003e =34ppm\u003csub\u003ew\u003c/sub\u003e) under today’s (light color, mid-day 20.5°C, 500 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 47 % WHCmax) and future (dark color, mid-day 23.9°C,\u0026nbsp; 800 ppm\u003csub\u003ev\u003c/sub\u003e CO\u003csub\u003e2\u003c/sub\u003e, 45 % WHCmax) climatic conditions. PERMANOVA was used to analyze Bray-Curtis dissimilarity matrices, comparing the effects of soil-spinach combinations, climate impacts, and their interaction across three biological replicates. (B) Heatmap showing differences in the relative abundance of OTUs significantly influenced by climate and Cd or Zn concentrations in spinach leaves under future versus today’s climatic conditions, along with their potential functionality. For OTU functionality assignments, refer to Table S8. The p-values are represented as follows: \"***\" = p \u0026lt; 0.001, \"**\" = p \u0026lt; 0.01, \"*\" = p \u0026lt; 0.05, and \"NS\" = p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/170f177af31a3d8f14ec046d.png"},{"id":75396465,"identity":"7c4ab8ae-764d-4c66-9e1b-42ba1341d2fa","added_by":"auto","created_at":"2025-02-04 06:51:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1777958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/b76f4ae9-61ef-4618-8ed2-276889d93fe7.pdf"},{"id":75395480,"identity":"82d994c9-7a4d-48fd-8919-a407fec82e96","added_by":"auto","created_at":"2025-02-04 06:27:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2856329,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement_Pienkowska_et_al\u003c/p\u003e","description":"","filename":"ClimateMetalSpinachPienkowskaetalSupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-5947512/v1/1d49da36781b5ef69babfcf2.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eClimate change increases toxic cadmium loads more than nutritional metals in spinach\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe quality of food is intricately linked to the presence and amounts of micronutrients and contaminants, holding relevance for human health\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Foods rich in essential micronutrients like zinc (Zn), magnesium (Mg), or manganese (Mn) are vital for supporting various physiological functions including immune function, fertility, muscles and bone growth\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, current global food systems face challenges in providing adequate micronutrients with, for example, 17% of the global population deficient in dietary Zn\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The intensification of agriculture since the 1960s has led to higher biomass yields but caused micronutrient dilution in crops\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Additionally, non-essential metals such as cadmium (Cd), can accumulate in crops, posing significant health risks to consumers, including carcinogenic effects and damage to the kidneys and skeletal system\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNutritional and contaminant metals, occur naturally in agricultural soils, but their concentrations and mobility are influenced by human activities such as farming, industrial operations, and vehicle emissions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In Europe and the US, around 75% of topsoils contain moderate Cd levels, with some soils exceeding 1 mg Cd kg⁻\u0026sup1; dry soil still used for food production\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recently it was demonstrated that future climatic conditions may increase Cd mobility in agricultural soils\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. 4\u0026deg;C higher daytime temperatures, doubled atmospheric CO\u003csub\u003e2\u003c/sub\u003e and slightly lower soil moisture caused two times higher Cd concentrations in the pore water of these soils, which even transferred into the mobile fraction of the soil approximated with 0.01 M CaCl\u003csub\u003e2\u003c/sub\u003e extractions. This effect is strongest in soils with a pH\u0026thinsp;\u0026lt;\u0026thinsp;7, covering approximately 70% of global cropland\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, while soils with pH\u0026thinsp;\u0026gt;\u0026thinsp;7 showed no significant shifts due to Cd binding with carbonate minerals\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe observed increase in the concentration of Cd in the porewater due to climate change raises concerns about the potential transfer of this harmful metal into crops grown in oxic soil systems, posing a threat to human nutrition. This phenomenon has already been demonstrated for rice grown in flooded soils under greenhouse conditions, where future climatic conditions increased the bioavailability of the most prominent contaminant in rice systems, arsenic, ultimately decreasing rice yields and increasing regulatory inorganic grain arsenic limits\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Such links between climate change, metallic contaminant, or micronutrient phytoavailability in oxic soils - approximately 90% of agricultural soil\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e-remain understudied.\u003c/p\u003e \u003cp\u003eElevated atmospheric CO\u003csub\u003e2\u003c/sub\u003e and temperature have differential effects on plant growth and performance. Increasing atmospheric CO\u003csub\u003e2\u003c/sub\u003e stimulates the photosynthetic rates, increasing water and nutrient use efficiency and allocation of more photosynthetic product to the root, ultimately resulting in greater biomass and increased yields\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. As temperature rises, photosynthetic rates and yields increase up to the plant's optimum, but excessive heat can reduce yields and offset the positive effects of elevated CO₂\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we examine for the first time the climate change induced transfer of harmful Cd and nutritional Zn into spinach, a leafy vegetable contributing 32\u0026nbsp;million tons annually to global food production\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, which is grown on oxic soils and known for accumulating Cd in edible parts\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our findings shed light on the potential risks posed by metal contamination to crops in the future and highlight the importance of mitigating these risks for food security and quality.\u003c/p\u003e \u003cp\u003eFor this study, we utilized four geochemically diverse agricultural soils from different areas in Germany and four commonly cropped spinach varieties to examine the potential compounding impacts of a changing climate on soil metal fate. Natural soil Cd and Zn concentrations ranged from background to contaminated\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (Figure S1A). For today\u0026rsquo;s climate variables, we examined 20\u0026deg;C midday temperatures, ambient atmospheric CO\u003csub\u003e2\u003c/sub\u003e, and 45% soil water holding capacity, which represent climatic conditions commonly used for spring and fall cultivation of these spinach varieties according to suppliers\u0026rsquo; recommendation. Future climatic conditions applied in this study are compared to today's baseline and included a 3.4\u0026deg;C temperature rise, a 290 ppm\u003csub\u003ev\u003c/sub\u003e atmospheric CO₂ increase, and a 2-percentage-point decrease in soil moisture relative to water-holding capacity. These conditions align with the projected climate scenario under Shared Socioeconomic Pathway SSP2\u0026ndash;4.5\u003csup\u003e19\u003c/sup\u003e, identified as the most likely scenario for the year 2100 relative to today\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and a SSP4-8.5 scenario relative to pre-industrial times.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDeterminants of edible spinach yield and quality\u003c/h2\u003e \u003cp\u003eCadmium accumulated in edible leaves of the Schlunzig-Lazio, Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador, Schladebach-Corvair combinations to 1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11, 3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, 0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;g Cd g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry leaf, respectively, under today\u0026rsquo;s climatic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A shift to future climatic conditions statistically increased leaf Cd for Schlunzig-Lazio by 54% to 2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Gro\u0026szlig;schirma-Butterflay by 27% to 4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T\u0026uuml;bingen-Metador by 30% to 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and Schladebach-Corvair by 26% to 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;g Cd g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry leaf. Root Cd concentrations were 1.1-6.9-times higher than in leaves under today's climatic conditions, depending on the soil-spinach combination. Under future climatic conditions, root Cd increased by 26\u0026ndash;54%, with a significant increase for the Gro\u0026szlig;schirma-Butterflay combination (Table S1A).\u003c/p\u003e \u003cp\u003eThe micronutrient Zn accumulated to 75\u0026thinsp;\u0026plusmn;\u0026thinsp;4, 92\u0026thinsp;\u0026plusmn;\u0026thinsp;14, 67\u0026thinsp;\u0026plusmn;\u0026thinsp;3 and 53\u0026thinsp;\u0026plusmn;\u0026thinsp;5 \u0026micro;g Zn g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry leaf in edible leaves of the Schlunzig-Lazio, Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador, Schladebach-Corvair combinations, respectively, under today\u0026rsquo;s climatic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Future conditions statistically increased leaf Zn in Schlunzig-Lazio by 45% to 109\u0026thinsp;\u0026plusmn;\u0026thinsp;8 Zn g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry leaf. Edible leaf Zn were not statistically different under future conditions in Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador, Schladebach-Corvair combinations compared to today's climate and were 139\u0026thinsp;\u0026plusmn;\u0026thinsp;21, 67\u0026thinsp;\u0026plusmn;\u0026thinsp;3 and 53\u0026thinsp;\u0026plusmn;\u0026thinsp;3 \u0026micro;g Zn g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry leaf, respectively. Root Zn was 0.7-times lower than in leaves for Gro\u0026szlig;schirma-Butterflay under today's climatic conditions, and 2.0-2.8-times higher for the other three soil-spinach combinations. Under future conditions, root Zn increased by 10\u0026ndash;60%, with a significant increase for the Gro\u0026szlig;schirma-Butterflay combination (Table S1A). There was a positive correlation between leaf Cd and Zn concentration in all soil-spinach combinations under both climatic conditions (Figure S2). Concentrations of two other micronutrients, Mn and Mg, increased in leaves and roots under future climatic conditions, sometimes significantly (Table S1A,B).\u003c/p\u003e \u003cp\u003eDry leaf yields increased significantly for Schlunzig-Lazio by 36%, Gro\u0026szlig;schirma-Butterflay by 56%, T\u0026uuml;bingen-Metador by 192% and not significantly for Schladebach-Corvair by 16% under future climatic conditions (Figure S3A). Future climatic conditions significantly increased total Cd and Zn accumulated by spinach for Schlunzig-Lazio, Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador and not significantly for Schladebach-Corvair (Figure S3B,C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Cd soil-to-root transfer factor was 6-100-times higher than the Cd root-to-shoot translocation factor, depending on the soil-spinach combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under today\u0026rsquo;s conditions, the Cd transfer factors for Schlunzig-Lazio, Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador, Schladebach-Corvair were 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, 6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, 8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 and 20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3, respectively. Future climatic conditions increased Cd transfer factors in all combinations, by 23% for Schlunzig-Lazio (8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0), 37% for Gro\u0026szlig;schirma-Butterflay (8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, significant), 70% for T\u0026uuml;bingen-Metador (14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2), and 56% Schladebach-Corvair (31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1). The Cd root-to-shoot translocation factors under today\u0026rsquo;s climatic conditions were 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 for Schlunzig-Lazio, T\u0026uuml;bingen-Metador, and Schladebach-Corvair, and 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for Gro\u0026szlig;schirma-Butterflay. Future conditions increased the translocation factor by 50% to 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 for Schlunzig-Lazio and T\u0026uuml;bingen-Metador. It remained 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 for Schladebach-Corvair, but decreased by 9% to 0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for Gro\u0026szlig;schirma-Butterflay. The Cd bioconcentration factors increased in all soil-spinach combinations under future climatic conditions, with significant increases for Schlunzig-Lazio and Gro\u0026szlig;schirma-Butterflay (Table S2).\u003c/p\u003e \u003cp\u003eThe Zn soil-to-root transfer was 0.35-8-times higher than the Zn root-to-shoot translocation factor, depending on the soil-spinach combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Under today\u0026rsquo;s conditions, the Zn transfer factors for Schlunzig-Lazio, Gro\u0026szlig;schirma-Butterflay, T\u0026uuml;bingen-Metador, Schladebach-Corvair were 0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0, 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, respectively. Future conditions increased Zn transfer factors in all combinations, by 11% for Schlunzig-Lazio (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1), by 50% for Gro\u0026szlig;schirma-Butterflay (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, significant), by 22% for T\u0026uuml;bingen-Metador (2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7), and 57% for Schladebach-Corvair (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7). The Zn root-to-shoot translocation factors under today\u0026rsquo;s climatic conditions were 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for Schlunzig-Lazio, 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 for Gro\u0026szlig;schirma-Butterflay, 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 for T\u0026uuml;bingen-Metador, 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for Schladebach-Corvair. Future conditions increased the translocation by 20% to 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for Schlunzig-Lazio and by 50% to 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 for T\u0026uuml;bingen-Metador. It did not change for Gro\u0026szlig;schirma-Butterflay and Schladebach-Corvair. The Zn bioconcentration factor increased in all soil-spinach combinations under future climatic conditions, except for the T\u0026uuml;bingen-Metador combination, with a significant increase observed in the Schlunzig-Lazio combination (Table S2).\u003c/p\u003e \u003cp\u003eThe Gro\u0026szlig;schirma-Butterflay combination reached the seed-producing development stage and future climatic conditions only slightly accelerated spinach development for all soil-spinach combinations (Figure S4A). Chlorophyll a and hydrogen peroxide concentrations in leaves, used here as approximation for plant health, were not affected by future climatic conditions (Figure S4B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoil biogeochemical determinants of spinach metal uptake\u003c/h3\u003e\n\u003cp\u003eTo minimize disturbance of spinach growth and root system integrity, time-series data of rhizosphere geochemical dynamics were collected only for the Gro\u0026szlig;schirma-Butterflay combination. Rhizosphere exchangeable Cd, approximated with 0.01 M CaCl\u003csub\u003e2\u003c/sub\u003e, tended to be 21% higher under future conditions compared to today throughout the growing period, excluding the starting point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Climate-associated differences in CaCl\u003csub\u003e2\u003c/sub\u003e-extractable Zn became evident from day 28 onward, with Zn tending to be higher under future conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Rhizosphere pH consistently tended to be lower under future conditions compared to today, with a 0.2-unit decrease by day 55. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Rhizosphere electrical conductivity tended to be higher under future conditions compared to today throughout the growing period reaching a 71% increase by day 55 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At day 55, rhizosphere 16S rRNA gene and transcript copy numbers were not statistically 5.3- and 2.9-fold higher, respectively, under future conditions compared to today\u0026rsquo;s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE,F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRhizosphere soil was sampled at harvest in three soil-variety combinations not disturbed by times-series sampling. Water-extractable TC ranged from 88\u0026ndash;155 mg g⁻\u0026sup1; dry soil under today's climate, varying by combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Under future conditions, TC increased by 3\u0026ndash;45%, with a significant risein T\u0026uuml;bingen-Metador. Rhizosphere soil TOC ranged from 69\u0026ndash;131 mg g⁻\u0026sup1; under today's climate, increasing by 7% for Schlunzig-Lazio, 50% for T\u0026uuml;bingen-Metador, and showing no change for Schladebach-Corvair. Rhizosphere soil IC ranged from 11\u0026ndash;24 mg g⁻\u0026sup1; under today's climate, rising by 12\u0026ndash;18% across combinations under future conditions. Rhizosphere TN ranged from 13\u0026ndash;21 mg g⁻\u0026sup1; under today's climate, increasing by 6% for Schlunzig-Lazio, 5% for T\u0026uuml;bingen-Metador, and remained unchanged for Schladebach-Corvair. Using the same extracts, 50 metabolites were measured and categorized to metabolite classes (Table S3-S7). Log2-fold-changes between climates are presented, with values above 1 indicating increased metabolite concentrations under future conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Responses varied by combination, except for metal chelators, which consistently increased: 1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92-log2-fold in Schlunzig-Lazio, 2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41-log2-fold in T\u0026uuml;bingen-Metador, and 3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51-log2-fold in Schladebach-Corvair. Other responses were combination-specific: amino acids rose by 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37-log2-fold in Schlunzig-Lazio, fermentates by 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30-log2-fold and pathogen suppressors by 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24-log2-fold in T\u0026uuml;bingen-Metador, and Krebs cycle intermediates by 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37-log2-fold in Schladebach-Corvair.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRoot-associated microbiome soil was sampled at harvest in three soil-variety combinations not disturbed by time-series sampling. Bacterial 16S rRNA gene copy numbers, alpha diversity, richness, and evenness varied by soil-spinach combination. Future climatic conditions tended to increase these metrics for Schlunzig-Lazio and T\u0026uuml;bingen-Metador but decreased them for Schladebach-Corvair (Figure S5A,B). Root-associated bacterial community structure clustered primarily by soil-spinach combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), explaining 60% of the variation (PERMANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Climatic conditions caused distinct shifts in community structure, accounting for 10% of the variation (p\u0026thinsp;=\u0026thinsp;0.002), while their interaction with soil-spinach combinations an additional 8% (p\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e \u003cp\u003eWithin the root-associated bacterial community, twelve OTUs were statistically associated with climate and at least one metal (Cd or Zn) concentration in spinach leaves, using a threshold of α\u0026thinsp;=\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Eight OTUs were identified in the Schlunzig-Lazio combination, all correlated with both Cd and Zn leaf concentrations. Of these, five OTUs (\u003cem\u003eDokdonella, Terrimonas, Chitinophagaceae, Georgfuchsia, Ellin6055\u003c/em\u003e) increased in relative abundance, while three OTUs (\u003cem\u003eA4b, Sphingopyxis, Nannocystis\u003c/em\u003e) decreased. The Schladebach-Corvair combination was the second most affected, with three OTUs (\u003cem\u003eAcidibacter, Fibrobacteraceae, Sandaracinus\u003c/em\u003e) increasing in relative abundance under future conditions. All three correlated with leaf Cd concentration, with \u003cem\u003eFibrobacteraceae\u003c/em\u003e also correlating with Zn. Lastly, in the T\u0026uuml;bingen-Metador combination, only one OTU (\u003cem\u003eFlavobacterium\u003c/em\u003e) decreased in relative abundance under future conditions and was solely correlated with Cd concentrations in spinach leaves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFuture climate conditions in this study were compared to today's baseline and included a 3.4\u0026deg;C temperature rise, a 290 ppm\u003csub\u003ev\u003c/sub\u003e atmospheric CO₂ increase, and a 2-percentage-point reduction in soil moistureࣧthe most likely scenario for the year 2100 relative to today\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These conditions fostered favourable growth for spinach, leading to yields increases of 16\u0026ndash;192%, depending on the soil-spinach combination, compared to today\u0026rsquo;s climate (Figure S3A). Elevated atmospheric CO\u003csub\u003e2\u003c/sub\u003e and temperature up to the plant's optimum, combined with adequate water availability, enhance photosynthesis and biomass production\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, further temperature increases, reduced soil moisture, or the use of spinach varieties with different environmental optima may reduce yields\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClimate change impacts spinach beyond yields, affecting edible product quality, including nutrient and toxin levels. Previous reports raised concerns that CO\u003csub\u003e2\u003c/sub\u003e-driven plant biomass increases could dilute micronutrient concentrations in yields due to limited soil availability and tight nutrient uptake controls\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, most studies focused on elevated CO\u003csub\u003e2\u003c/sub\u003e alone. Recent experiments combining temperature and CO\u003csub\u003e2\u003c/sub\u003e increases reveal a more complex relationship. Some studies suggest higher temperatures counteract CO\u003csub\u003e2\u003c/sub\u003e-induced micronutrient dilution in crops like soybean, wheat, and rice, likely through transpiration-driven increases in nutrient mass flow\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To our knowledge, this is the first study to explore these effects in leafy vegetables. Our finding supports that temperature helps maintain, or in some soil-spinach combinations, even increases concentrations of essential nutrients Zn, Mn, and Mg, under future climates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table S1).\u003c/p\u003e \u003cp\u003eIn contrast, harmful Cd accumulation increased by 25\u0026ndash;50% under future conditions across all combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This greenhouse study provides the first evidence that future climatic conditions enhance metal transfer from oxic soils to crops. Climate-induced Cd accumulation in spinach leaves may result from changes in its soil mobility, soil-to-root transfer, and root-to-shoot translocation, discussed in the following sections.\u003c/p\u003e \u003cp\u003eFor Cd uptake in spinach, it must first be converted into phytoavailable forms, such as dissolved ions or weakly adsorbed species on soil particles. Climate-driven increases in metal mobility have been shown in other systems, such as arsenic in rice rhizospheres, where redox processes, particularly microbially mediated reductive dissolution of iron and arsenic, enhance mobility in flooded, anoxic paddy soils\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, these redox processes are less relevant for oxic soils and non-redox-active Cd. A recent study by Drabesch et al. using similar climatic variables to those applied here demonstrated that future climatic conditions influence Cd dynamics in oxic soils by altering soil microbiomes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Specifically, ammonium oxidation stimulated by climate-induced shifts lowered soil pH, displacing Cd through proton activity, in Drabesch et al. observed in T\u0026uuml;bingen soil\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, here confirmed in Gro\u0026szlig;schirma soil. Acidification dissolves Cd co-precipitates, increases soil particle surface charge, and reduces Cd binding via electrostatic repulsion\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In support of Drabesch et al\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we also found taxa-based evidence for enhanced N cycling in the Schlunzig-Lazio combination, the soil with the highest total Cd content. Two root-associated taxa (\u003cem\u003eDokdonella, Chitinophagaceae\u003c/em\u003e; Table S6) statistically correlated with both climate and Cd were identified as potential nitrifiers, and showed increased abundance under future conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Increased N pools, particularly amino acids in the rhizosphere (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), may stem from plant defence mechanisms against Cd\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, circumstantially providing substrates for ammonium oxidation and further mobilizing Cd. Unlike Drabesch et al., who excluded plants in their study and observed no differences in dissolved carbon\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we found a tendency for climate-induced increases in rhizosphere water-extractable carbon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This is likely driven by enhanced photosynthesis-derived carbon transfer to the soil via root exudation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Spinach and soil microbiomes exhibited increased growth and activity under future climatic conditions (Figure S3; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), elevating nutrient demands. Both likely excreted metabolites to mobilize nutrients, including chelators, whose collective concentrations significantly increased across three soil-spinach combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, chelators may inadvertently mobilize Cd and not just nutrients\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, contributing to Cd\u0026rsquo;s increased soil mobility. This mechanism was crucial even in high-pH soils, for example, Cd accumulation in the Schladebach-Corvair combination occurred despite a soil pH of 7.6 (Figure S1). Moreover, while Drabesch et al. found future climatic conditions altered soil carbon composition toward larger, more oxidized, lower energy organic matter\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, our inclusion of plant inputs maintained fresh carbon sources (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), fostering favorable conditions for bacterial growth. Soil- and root-associated bacterial abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; Figure S5A) and activity increased, evidenced by higher 16S rRNA transcript numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and elevated Krebs cycle metabolites in the soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the T\u0026uuml;bingen-Metador combination, elevated fermentative metabolites suggested organic matter turnover was so intense that anoxic microenvironments may have formed\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Enhanced bacterial metabolism may have increased soil carbon turnover and decomposition, raising electrical conductivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and further mobilizing Cd. Organic acids from carbon sources, Krebs cycle products, and fermentative metabolites (Table S3-S7) lowered soil pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) with impacts on Cd mobility as discussed above. Soil Cd mobility increased earlier and more significantly than Zn during the growth period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B), resulting in higher Cd than Zn accumulation in spinach leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). This aligns with previous findings, as Zn\u0026rsquo;s lower first hydrolysis constant and larger hydrated radius increase its tendency to adsorb and precipitate in soils compared to Cd\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to altering metal accumulation in spinach, climatic condition and potentially climate-enhanced soil metal mobility shifted root-associated microbiome composition, potentially affecting long-term soil fertility, plant performance, and ecosystem functioning\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The Schlunzig-Lazio combination, with the highest total soil Cd and Zn (Figure S1A) and the only combination where leaf Zn increased under future conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), exhibited the most root-associated taxa significantly correlated with climate and phytoavailable Cd and Zn (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In this soil, climate-enhanced metal mobility may reach stress-inducing levels, evidenced by increased abundances of metal-resistant and metal-immobilizing taxa (\u003cem\u003eGeorgfuchsia\u003c/em\u003e, Ellin6055, \u003cem\u003eTerrimonas\u003c/em\u003e; Table S6). Conversely, negatively affected taxa included complex organic carbon degraders (A4b, \u003cem\u003eSphingopyxis, Nannocystis\u003c/em\u003e; Table S6), suggesting metal toxicity shifts the microbiome toward oligotrophs, potentially disrupting future soil nutrient cycling. The second most affected root-associated community was in the Schladebach-Corvair combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Despite low soil Cd and Zn (Figure S1A), increased metal mobility may not yet be toxic but stimulatory, driving microbes to seek additional energy sources for resistance\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This likely promoted an increase in complex organic carbon degraders and metal-resistant taxa (\u003cem\u003eAcidibacter, Fibrobacteraceae, Sandaracinus\u003c/em\u003e; Table S6). Lastly, the least affected root-associated community was in the T\u0026uuml;bingen-Metador combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), which had the second-lowest total metal concentrations (Figure S1A) and the highest carbon and nitrogen pools (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This nutrient abundance likely supported bacterial stability, minimizing community changes. However, the only taxon significantly affected by climate-enhanced Cd mobility was the plant-growth promoter \u003cem\u003eFlavobacterium\u003c/em\u003e (Table S6), suggesting its potential vulnerability to future Cd mobilization.\u003c/p\u003e \u003cp\u003eOnce mobilized, metal cations move towards the roots via diffusion and advection. Diffusion occurs as cations are attracted to the negatively charged root surface, creating a concentration gradient, while advection transports ions with water moving toward roots due to plant transpiration\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Future conditions likely enhanced advective transport as elevated atmospheric CO₂ increases stomatal opening, leading to greater water evaporation from leaves, further amplified by higher temperatures\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Cadmium, being more mobile in soil solution than Zn (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B), likely adsorbed more readily to root surfaces, with increased transpiration boosting passive Cd uptake relative to Zn\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As an essential micronutrient, Zn uptake exceeds passive processes, requiring active transport mechanisms like ZIP family proteins in spinach roots\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, due to its chemical similarity to Zn, Cd can also be mistakenly transported via Zn transporters\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, likely intensified by its increased soil mobility. Under excess metal concentrations in root cell cytosol, homeostatic mechanisms regulating Zn and Cd involve efflux into compartments like the apoplast, vacuole, and Trans-Golgi Network\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, immobilizing metals to reduce toxicity but retaining them in root tissues. Overall, future climatic conditions enhanced soil-to-root transfer for both Cd and Zn, with Cd transfer factors increasing by 46% on average compared to 36% for Zn across soil-spinach combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C).\u003c/p\u003e \u003cp\u003eMetals taken up by roots are transported to aerial parts via the xylem, driven by transpiration and mediated by metal transporters. This tightly regulated root-to-shoot translocation varies among spinach varieties, influencing final leaf Zn and Cd concentrations\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Zinc root-to-shoot translocation factors tended to increase under future climatic conditions in only half the tested combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Despite climate-induced biomass increases, leaf Zn concentrations remained stable in most varieties, reflecting robust variety-specific Zn homeostasis. Only the Schlunzig-Lazio combination showed a climate-induced increase in leaf Zn (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), possibly due to high Zn in Schlunzig soil (Figure S1A), which, when mobilized under future climate, may have exceeded Lazio's Zn immobilization capacity. If Cd is not immobilized in roots, it moves to shoots exploiting Zn transport pathways or passive diffusion along transpirational fluxes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Under future conditions, Cd translocation to shoot increased more than Zn, with a 40% average rise in root-to-shoot translocation factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This matches the 46% rise in Cd soil-to-root transfer factors, confirming spinach efficiently transports Cd to shoots\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and highlighting the soil-to-root barriers importance, as spinach can enhances leaf translocation without signs of toxicity (Figure S4).\u003c/p\u003e \u003cp\u003eResearch on climate change impacts on food security has traditionally focused on yield quantity. Here, we demonstrated for the first time that Cd accumulation by plants, a non-redox-active metal and major contaminant in oxic soils (90% of agricultural soils\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e), is also affected by future climatic conditions. This was not matched by equal increases in essential nutrients like Zn, Mn, and Mg, which varied depending on the plant-soil combination. Under the experimental conditions, future climate could increase Cd accumulation in spinach, potentially raising consumer exposure to harmful Cd levels and posing financial risks to agriculture due to safety standard non-compliance. These findings likely extend to other leafy vegetables and warrant further study on staple grain crops like wheat. Our results show that future climates not only boost Cd transfer from soil to roots but also enhances its translocation to leaves. Mitigation strategies such as soil management techniques that immobilize Cd more effectively than Zn (e.g. organic amendments\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e) and breeding low-Cd spinach varieties\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e could help address these risks.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003e \u003cb\u003eSoil and spinach characterization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo ensure that the findings in this study are applicable to a wide range of soils and spinach variety, four unique combinations of soil types and spinach varieties were selected for this study (Figure S1). Four agricultural soils, originating from distinct geographic locations and therefore of different geochemical properties, were used. They are denoted based on their geographic origins within Germany as following: Schlunzig (50\u0026deg;47'29.3\"N 12\u0026deg;30'02.6\"E), Gro\u0026szlig;schirma (50\u0026deg;57'42.7\"N 13\u0026deg;16'44.6\"E), T\u0026uuml;bingen (48\u0026deg;32'48.0\"N 9\u0026deg;02'30.7\"E), and Schladebach (51\u0026deg;18'30.6\"N 12\u0026deg;06'16.4\"E). Soils were consistently collected from the top 20 cm, mixed well, air-dried, sieved through a 4 mm mesh sieve, and stored in the dark. The time of sampling and geochemical characteristics of each soil are summarized in Table S7. Unless otherwise stated in the cited sources, the following protocols were used for soil characterization. Texture was determined in triplicates by a hydrometer in sodium hexametaphosphate solution. The fraction of sand was quantified with a hydrometer after 40 s, silt after 2 h, and the remaining fraction was calculated as clay. Soil pH was quantified in triplicates from air-dried soil with 0.01 M CaCl\u003csub\u003e2\u003c/sub\u003e at a 1:5 w/v ratio after 24 h at room temperature. The cation exchange capacity was quantified in triplicates from air-dried soils with 0.1 M BaCl\u003csub\u003e2\u003c/sub\u003e (analytical grade) at a 1:25 w/v ratio for 4 h of shaking (200 rpm) at room temperature. The total carbon and nitrogen contents were quantified in triplicates from 40\u0026deg;C-dried soils by combustion in tin foil balls (Flash 2000 Organic Elemental Analyzer, Delta V Advantage Isotope Ratio MS). The elemental contents of the soils were quantified in 10 mm thick soil powders dried at 105\u0026deg;C using X-ray fluorescence (XRF, SPECTRO XEPOS spectrometer, SPECTRO Analytical Instruments GmbH, Germany). To maximize Cd quantification, region 2 between 6 and 15 keV was extended to 900 ms. Four spinach varieties commonly used in Europe were selected for this study: Lazio, Butterflay, Metador, Corvair (Table S7). The seeds were stored in the dark at room temperature until the experiment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGreenhouse setup and pot study design.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Gro\u0026szlig;schirma soil \u0026ndash; Butterflay spinach combination was grown in Spring 2023 and the other three soil-spinach combinations in Autumn 2023 in daylight-fed greenhouses at the UFZ Research Station in Bad Lauchst\u0026auml;dt (51\u0026deg;23'33.6\"N 11\u0026deg;52'33.6\"E). For today\u0026rsquo;s climate variables, we examined midday temperatures of 20\u0026deg;C and ambient atmospheric CO\u003csub\u003e2\u003c/sub\u003e, which represent commonly used for spring and fall cultivation of the used spinach varieties according to suppliers\u0026rsquo; recommendation (Table S8). Future climatic conditions applied in this study are compared to today's baseline and included a 3.4\u0026deg;C temperature increase, a 290 ppm\u003csub\u003ev\u003c/sub\u003e rise in atmospheric CO₂. These conditions align with the projected climate scenario under Shared Socioeconomic Pathway SSP2\u0026ndash;4.5\u003csup\u003e19\u003c/sup\u003e, identified as the most likely scenario for the year 2100 relative to today\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and a SSP4-8.5 scenario relative to pre-industrial times. To ensure chamber-independent results, three different chambers were used and their climatic conditions were rotated between experimental runs.\u003c/p\u003e \u003cp\u003eBlack plastic pots (9x9x20 cm) were filled with ~\u0026thinsp;1.4 kg of air-dried soil. The first irrigation was carried out with tap water by capillarity overnight. After reaching saturation, four spinach seeds that had been soaked for two days (Gro\u0026szlig;schirma- Butterflay combination) or one day (all other soil-spinach combinations) in autoclaved Milli-Q\u0026reg; water were planted in pots 1 cm deep and 1.5 cm apart. Young seedlings were thinned to 2 plants (Gro\u0026szlig;schirma- Butterflay combination) or 1 plant (all other soil-spinach combinations) within the first 2 weeks. Irrigation, conducted twice a week, remained consistent across both climate conditions and resulted in an average of 47% WHC under today's climatic conditions. However, due to higher atmospheric temperatures in the future climate scenario, soil moisture levels were lower, averaging 45% WHC (Figure S6).\u003c/p\u003e \u003cp\u003eEach soil-spinach variety combination in each climate was carried out with twelve replicates using a randomized block design\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To avoid biases in light exposure, atmospheric temperature, and drafts in the growth chambers, the blocks were moved weekly.\u003c/p\u003e \u003cp\u003eTime-resolved soil geochemical data were obtained for five to seven of the twelve replicates of one of the Gro\u0026szlig;schirma-Butterflay soil-variety combination to minimize disturbance to spinach growth and maintain roots systems integrity in the other varieties and replicates. Soil samples were collected to monitor CaCl\u003csub\u003e2\u003c/sub\u003e-extractable Cd and Zn, pH, and electrical conductivity. Five-cm deep soil cores were taken on days 1, 8, 29 and 55, randomly rotated among replicates of the Gro\u0026szlig;schirma-Butterflay combination so that each replicate was sampled twice during the experiment. Cores were thoroughly mixed, aliquoted, flash-frozen in dry ice, and stored at -20\u0026deg;C until analyses. At harvest, rhizosphere soil samples for bacterial analysis were collected from three replicates using the same method and stored at -80\u0026deg;C for subsequent analyses (see bacterial analysis section). For rhizosphere soil solution carbon profiling, soil samples were collected at harvest from the three remaining soil-variety combinations that had not been disturbed by time-series sampling. Horizontal cores were taken at a depth of 10 cm to obtain rhizosphere soil at harvest. These cores were thoroughly mixed, roots removed, aliquoted, flash-frozen in dry ice, and stored at -20\u0026deg;C for water-extractable root exudate, carbon and nitrogen analysis (see soil analysis section).\u003c/p\u003e \u003cp\u003ePlant-related analyses were done for all replicates. Plant growth and development were tracked weekly over the duration of cultivation. At time of harvest, spinach plants were first photographed (Figure S1B). To minimize photosynthesis-related artifacts in plant and soil data, sampling was organized in blocks, and done in parallel per climate. Edible yields were quantified by wet and dry mass, washed in deionised water, and flash-frozen in dry ice until storage at -80\u0026deg;C before analysis of metal contents and plant health (see plant analysis section). Roots were separated from soil, washed thoroughly in deionised water, weighed and flash-frozen in dry ice until storage at -80\u0026deg;C before analysis of metal contents (all replicates) and root-associated bacteria (three replicates from the three soil-variety combinations that were not disturbed by time-series soil sampling).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlant analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEdible yields and root mass were normalized to their dry weight (40\u0026deg;C-dried until constant weight). For metal and plant health quantification, spinach was ground in liquid nitrogen. Metal contents of edible yields and roots were assessed from 12 biological replicates by extracting approximately 0.1 g biomass in 3 mL of 65% nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e, analytical grade) and 2 mL of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (analytical grade) in a microwave digester (Mars6, CEM, USA) with a 15-min ramp phase to 15 min at 180\u0026deg;C, followed by a 30 min cool-down phase\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Extractants were filtered through filter paper (Whatman qualitative filter paper, Grade 1) and diluted in MQ water. Extraction blank and a reference material (European Reference Material ERM\u0026reg;-CD281) were extracted along with each extraction run for later computational comparison and alinement between runs. Elemental contents were quantified in leaf extraction filtrates with XXX. Leaf chlorophyll a was quantified by extracting ground, frozen leaves with 80% acetone and measuring absorbances spectrophotometrically at 663 and 645 nm. To evaluate climate stress in spinach, H₂O₂ contents were determined from 0.1% (w/v) trichloroacetic acid (TCA) extracts of frozen, ground leaf tissues at a 1:7.5 w/v ratio. Subsequently, 300 \u0026micro;L of 10 mM potassium phosphate buffer (pH 7) and 600 \u0026micro;L of 1 M potassium iodide were added to 300 \u0026micro;L of the 0.1% TCA extract. The mixture was incubated in the dark for 20 minutes, and absorbance was measured at 390 nm. H₂O₂ content was calculated using a standard curve.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSoil analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMobile Cd and Zn in soil were approximated using six biological replicates by a 15 minute extraction with 0.01 M CaCl\u003csub\u003e2\u003c/sub\u003e at a 1:6.67 w/v ratio at room temperature\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Extractants were filtered through 0.22 \u0026micro;m filters (Minisart\u0026reg; high flow, Sartorius, Germany), which were pre-washed with MQ water (to remove filter-associated organic matter impurities). Afterward, they were acidified with HNO\u003csub\u003e3\u003c/sub\u003e to a concentration of 2% and stored at 4\u0026deg;C until quantification by ICP-MS and ICP-OES as described above for plant material. Electrical conductivity and soil pH were measured using five-seven replicates at a fresh soil to ultra-pure water ratio of 1:5 (w/v). Electrical conductivity was determined after in the settling solution, and soil pH after 24 hours (SD335 Multi-Parameter Meter, Lovibond, Germany). Carbon and nitrogen profiles were extracted using 4 biological replicates by a 4 hour extraction with ultra-pure water at a 1:8 w/v ratio at room temperature\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Extractants were filtered as described for CaCl\u003csub\u003e2\u003c/sub\u003e extraction and stored at -20\u0026deg;C until analysis for total carbon (TC), total organic carbon (TOC), inorganic carbon (IC) and total nitrogen (TN) concentrations with a TOC analyzer (Multi N/C 2100S duo, Analytik Jena, Germany). The concentration of metabolites was measured using GC-MS (Shimadzu GC/MS TQ 8040, Japan) operated in Electron Ionisation mode (EI). Metabolites were measured either by headspace injection (MS acquisition mode: Scan) or as a liquid sample after derivatization (MS acquisition mode: MRM). For derivatization, samples were thawed on ice and 3000 pmol 13C-glucose was added as an internal standard to 250 \u0026micro;L sample prior to freeze-drying. The solids were re-dissolved with 50 \u0026micro;L methoxamine (20 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e pyridine), ultrasonicated for 10 min and subsequently incubated at 30\u0026deg; C for 90 min. Next, 70 \u0026micro;L N-methyl-N-(trimethylsilyl)-trifluor-acetamid (MSTFA) was added and samples were incubated at 40\u0026deg;C for 60 min, after which they were kept at room temperature for 2 h{Fiehn, 2000 #294}. A volume of 1 \u0026micro;L was used for injection in splitted mode on a Restek SH-Rxi-5SIL MS column (30 m; film 0.25 \u0026micro;m; diameter 0.25 mm). The injection port was heated to 280\u0026deg;C. For chromatographic separation a He flow rate of 1.1 mL min-1 and a temperature increase of 10\u0026deg;C min-1 from 100 to 320\u0026deg;C were employed. For the measurement via the headspace method, 250 \u0026micro;L of thawed sample together with 2500 pmol octanol as internal standard were added into 10 mL glass vials with closed lids. The total liquid amount is 1 mL containing 470 \u0026micro;L saltout media (882 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Ammonium sulfate and 238 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Sodium dihydrogen phosphate, 20 \u0026micro;L Phosphoric acid 85% and adjusted with MiliQ water to the end volume{Fiorini, 2016 #296}. After heating and shaking at 80\u0026deg;C for 30 min a volume of 1 mL of headspace gas was injected onto the column Stabilwax-DA (30 m; film 1 \u0026micro;m; diameter 0.32 mm) from Restek. The He flow rate was 1.1 mL min-1 and the column temperature increased by 10\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from 40 to 240\u0026deg;C{Zhang, 2018 #295}. Concentrations were quantified with a 8-point external calibration containing standards of all targeted analytes, ranging from 20 to max. 1.00E\u0026thinsp;+\u0026thinsp;07 pmol per 250 \u0026micro;L sample for headspace method and 20 to max. 3.00E\u0026thinsp;+\u0026thinsp;04 pmol per 250 \u0026micro;L sample for liquide sample method. Labsolutions Insight (Shimadzu, Japan) GCMS software was used for peak integration which was double checked manually. Relative peak areas and amount of metabolites were calculated with R using a self programmed shinyapp in Rstudio{Chang, 2025 #297}. Final metabolite data were annotated by class (Table S3-S5), with log2 fold changes between climate scenarios calculated for each class and block, followed by averaging these changes for each class and determining errors through propagation. All soil analyses were normalized to dry weight (105\u0026deg;C-dried until constant weight).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo analyze bacterial quantity and taxonomic diversity, nucleic acids were extracted following Lueders et al., 2004\u003csup\u003e40\u003c/sup\u003e, using 0.6 g of wet soil. The quality and quantity of DNA and RNA were verified by NanoDrop 2000c (ThermoFisher, USA) and fluorometric quantification with Invitrogen\u0026trade; Qubit\u0026trade; 3 Fluorometer Qubit\u0026reg; 3.0 Fluorometer (ThermoFisher, USA), respectively.\u003c/p\u003e \u003cp\u003eCopy numbers of the rhizosphere soil and root-associated bacterial 16S rRNA gene and transcript were amplified and quantified by qPCR on a CFX96\u0026trade; Real-Time PCR Detection System (Bio-Rad Laboratories, Germany). As a standard, the plasmid vector pCR2.1\u0026reg; (Invitrogen, Darmstadt, Germany) containing a cloned 16S rRNA gene fragment of \u003cem\u003eThiomonas\u003c/em\u003e sp. was used\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. A master mix was prepared with 1\u0026times; SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, United States), 75 nM of primer 341-F (5\u0026prime;-CCTACGGAGGCAGCAG-3\u0026prime;)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and 225 nM of primer 797-R (5\u0026prime;- GGACTACCAGGGTATCTAATCCTGTT-3\u0026prime;)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In a total volume of 10 \u0026micro;l, 1 \u0026micro;l DNA extract (500-fold diluted) or standard plasmid DNA (eight-fold dilution series) and 9 \u0026micro;l master mix were quantified in 96-well PCR plates (Bio-Rad Laboratories). The qPCR program was run with 3 min at 98\u0026deg;C, followed by 40 cycles of 15 s at 98\u0026deg;C and 30 s at 60\u0026deg;C. For verification, melting curve analysis was performed using CFX Manager\u0026trade; software. Data were obtained from biological triplicates, except for the Gro\u0026szlig;schirma-Butterflay combination, where one sample was lost, leaving only two replicates for analysis. Results were normalized to the dry weight of 105\u0026deg;C-dried soil.\u003c/p\u003e \u003cp\u003eRoot-associated bacterial 16S rRNA genes and transcripts were amplicon-sequenced using primers 515F and 806R\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The quality and quantity of the purified amplicons were assessed via agarose gel electrophoresis. Library preparation (Nextera, Illumina, USA) and sequencing were carried out using the 2\u0026times;250 bp MiSeq Reagent Kit v2 on an Illumina MiSeq sequencing system (Illumina, San Diego, USA). Bacterial raw reads quality-checked sequences and bioinformatics assembly of datasets were performed using a workflow primarily based on DADA2{Callahan, 2016 #298}. This was implemented through the standardized pipeline Dadasnake version 0.11{Wei\u0026szlig;becker, 2020 #299}. Primers were detected with a maximum allowance of 20% mismatch and were trimmed using Cutadapt version 1.18. Bacterial sequences were filtered with the following parameters: trunc_quality:13, trunc_length:170 bp for both forward and reverse and max_EE:0.2. Taxonomic assignment was conducted using the SILVA database v138.1{Quast, 2012 #162}. ASVs were clustered using VSEARCH with a threshold of 97%, resulting in operational taxonomic units (OTUs).\u003c/p\u003e \u003cp\u003eTo identify root-associated OTUs related to climate and Cd/Zn concentrations in spinach leaves, the following pipeline was applied: first, Spearman correlations were conducted to identify OTUs correlated with Cd or Zn concentrations. These OTUs were then tested using a t-test between current and future climatic conditions to determine those significantly affected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analyses, statistical evaluation and data visualization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe utilized the R programming language and conducted our analyses within the RStudio integrated development environment for statistical computing and graphics. Statistical analyses focused on comparing today versus future conditions within each soil-spinach combination, as comparing different soil-spinach combinations was not the goal of this study. T-tests and Welch\u0026rsquo;s tests (for unequal variances) were used for pairwise comparisons, while a linear mixed model with repeated measures assessed climate impacts on time-series data, with Tukey\u0026rsquo;s HSD (α\u0026thinsp;=\u0026thinsp;0.05) for post-hoc adjustments. Pearson correlation analyzed Cd and Zn concentrations in leaves within each soil-spinach combination. PERMANOVA assessed climate effects on root-associated bacterial community composition across all soils, as replicates were insufficient for analysis within individual soil-spinach combinations.\u003c/p\u003e \u003cp\u003eThree factors were calculated to estimate Zn and Cd mobilization within the soil-plant system. The bioconcentration factor (1) was calculated to estimate the transfer of metals from the soil into the total biomass of spinach plants.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{B}\\text{i}\\text{o}\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{f}\\text{a}\\text{c}\\text{t}\\text{o}\\text{r}=\\frac{\\text{S}\\text{h}\\text{o}\\text{o}\\text{t}+\\text{R}\\text{o}\\text{o}\\text{t}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{s}\\text{o}\\text{i}\\text{l}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe soil-to-root translocation factor (2) was calculated to estimate the transfer of metals from the soil specifically to the roots of spinach plants.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{o}\\text{i}\\text{l}\\:\\text{t}\\text{o}\\:\\text{r}\\text{o}\\text{o}\\text{t}\\:\\text{t}\\text{r}\\text{a}\\text{n}\\text{s}\\text{f}\\text{e}\\text{r}\\:\\text{f}\\text{a}\\text{c}\\text{t}\\text{o}\\text{r}=\\frac{\\text{R}\\text{o}\\text{o}\\text{t}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{s}\\text{o}\\text{i}\\text{l}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe root-to-shoot translocation factor (3) was calculated to assess the internal transfer of metals from the roots to the shoots within spinach plants\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{R}\\text{o}\\text{o}\\text{t}\\:\\text{t}\\text{o}\\:\\text{s}\\text{h}\\text{o}\\text{o}\\text{t}\\:\\text{t}\\text{r}\\text{a}\\text{n}\\text{s}\\text{l}\\text{o}\\text{c}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{f}\\text{a}\\text{c}\\text{t}\\text{o}\\text{r}=\\frac{\\text{S}\\text{h}\\text{o}\\text{o}\\text{t}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}{\\text{R}\\text{o}\\text{o}\\text{t}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS:\u003c/h2\u003e \u003cp\u003eThe authors have no competing interests, financial or non-financial, in relation to the work described within this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTION\u003c/h2\u003e \u003cp\u003eThis study was conceptualized by AP, AG and EMM. Experiments were planned by AP, AG with input from EMM, and plant growth support by IM. The experiments, plant maintenance, lab work and analysis were carried out by AP, AG, SK, PGR, NS, LDPS, EMM. Elemental measurements were carried out by MH. Metabolite measurements were carried out by JK and MS. Data interpretation was done by AP with primary input from EMM, TR, LDPS, MH and JK. The manuscript and supporting information were written by AP and EMM with additional input from all co-authors. Funding for the project was acquired by EMM.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS:\u003c/h2\u003e \u003cp\u003eWe thank Marie Mollenkopf, Carolina Vergara Cid, Karolin Seiferth, Hendrik Seifert, Nawshin Atia, Paula Gscheidel, Jennifer Horstmann, Aaron Jakob, Lieke Lipsch, Ayushi Parmar, Swati Sharma, Birgit Sawall for lab and greenhouse support. The microbial community composition data have been computed at the high-performance computing cluster EVE, a joint effort of both the Helmholtz-Center for Environmental Research-UFZ and the German Center for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig. We kindly thank the server admins. The Saxonian State Agency for Environment, Agriculture and Geology, especially Dorit Julich and Ingo M\u0026uuml;ller, and Eckart Wizemann for supply of soil. This work was financed through the Helmholtz Young Investigator Grant RhizoThreats.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY STATEMENT:\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are also available from the corresponding authors on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePinstrup-Andersen P (2009) Food security: definition and measurement. 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Appl Environ Microbiol 81:2173\u0026ndash;2181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuyzer G, Ramsing NB (1995) Molecular methods to study the organization of microbial communities. Water Sci Technol 32:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGensberger ET et al (2014) Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality. Water Res 67:367\u0026ndash;376\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaporaso JG et al (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 108, 4516\u0026ndash;4522\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Helmholtz Centre for Environmental Research","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"crops, heavy metal, nutrients, elevated temperature, elevated CO2, soil moisture","lastPublishedDoi":"10.21203/rs.3.rs-5947512/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5947512/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn addition to food quantity, food quality is paramount for meeting the demands of a growing global population. Food quality encompasses both nutritional and contaminant contents, yet their transfer within soil-crop systems remains poorly understood under impending climate change. This greenhouse study is the first to demonstrate that future climatic conditions increase the transfer of metals from oxic soils to crops, showcased for four soil-spinach variety combinations (\u003cem\u003eSpinacia oleracea\u003c/em\u003e). Future conditions raised harmful metal cadmium levels in edible spinach tissues by 26\u0026ndash;54%. In contrast, changes in micronutrient (Zn, Mn, Mg) contents were inconsistent and dependent on the specific soil-spinach combination. Climate-induced shifts in soil carbon composition and bacterial communities were linked to greater soil Cd phytoavailability, enhancing Cd transfer from soil to roots. These findings suggest that while spinach's nutritional values may remain stable, future conditions could lead to higher metal contaminants levels in edible tissues.\u003c/p\u003e","manuscriptTitle":"Climate change increases toxic cadmium loads more than nutritional metals in spinach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 06:27:26","doi":"10.21203/rs.3.rs-5947512/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c7244da-22bc-4e41-9024-9846911529d5","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-04T06:27:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 06:27:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5947512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5947512","identity":"rs-5947512","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}